Metabolism of circulating disaccharides in man and the rat.

The metabolism of circulating disaccharides was studied in adult humans and rats. After iv infusions of 10 g of either lactose, sucrose, or maltose in four adults, no rise in blood glucose was noted. A mean of 8.7+/-1.89 g of the lactose and 6.3+/-1.39 g of the sucrose was excreted in the 24-hour urine sample. Only 0.11+/-0.03 g of the infused maltose was recovered in the urine, suggesting that the maltose was metabolized.After injection of (14)C-labeled lactose and sucrose in rats, 6.2+/-2.7 and 7.6+/-2.4%, respectively, was oxidized to (14)CO(2) in 24 hours; 62.1+/-13.5 and 68.4+/-10.8% of the respective disaccharides was excreted into the urine. Conversely, after injection of (14)C-labeled maltose 54.6+/-7.0% was oxidized to (14)CO(2) and 4.8+/-3.9% excreted in the urine. The per cent of maltose oxidized to CO(2) was similar to that of glucose. In addition to small intestinal mucosa, homogenates of rat kidney, brain, and liver as well as serum were found to have measurable maltase activities. The role of these tissue maltases in the metabolism of circulating maltose and maltosyloligosaccharides is discussed.

Intestinal assimilation of a proline-containing tetrapeptide. Role of a brush border membrane postproline dipeptidyl aminopeptidase IV.

The mechanism of hydrolysis and absorption of a proline-containing tetrapeptide, Leu-Pro-Gly-Gly (10 mM) by rat intestine was examined in vivo by using jejunal perfusion methods. The peptide substrate and hydrolysis products were analyzed by use of an automated amino acid analyzer. Leucine, proline, and glycine were absorbed by the intestine at a significantly higher rate from the tetrapeptide than from an equivalent amino acid mixture. The analysis of the hydrolytic products in the lumen during in vivo perfusion of the tetrapeptide showed that two dipeptides, Leu-Pro and Gly-Gly, were the major products. These two dipeptides were also the major hydrolytic products when a purified rat intestinal brush border membrane preparation was incubated with Leu-Pro-Gly-Gly. The rate of hydrolysis of the tetrapeptide was much higher than that for several other proline-containing peptides (Leu-Pro, Pro-Leu, and Pro-Gly-Gly) that were tested. Studies using Gly-Pro-beta-naphthylamide, a specific substrate for postproline dipeptidyl aminopeptidase IV, showed that this enzyme is mainly localized to the brush border membrane and is responsible for the hydrolysis of the tetrapeptide into the two dipeptides Leu-Pro and Gly-Gly. Thus, brush border membrane dipeptidyl aminopeptidase IV very likely plays an important role at the intestinal mucosal cell surface in the final stages of digestion of proline-containing peptides.

Effect of growth and sodium butyrate on brush border membrane-associated hydrolases in human colorectal cancer cell lines.

The activities of brush border membrane-associated hydrolases such as alkaline phosphatase (Alkpase), aminopeptidase, dipeptidyl aminopeptidase IV (DAP-IV), sucrase, lactase, and trehalase were studied in 14 different human colorectal cancer cell lines. The effect of sodium butyrate, a known differentiating agent, and cell growth on the activities of these enzymes was also examined. All 14 cell lines exhibited brush border membrane enzyme activities, and in general, the activity of Alkpase, aminopeptidase, and DAP-IV was much higher than the disaccharidases. However, the specific enzyme activities varied among different cell lines. The induction of Alkpase activity by sodium butyrate occurred in most of the 14 cell lines (2- to 123-fold), while induction of the other enzyme activities was observed in several (1.5- to 3.5-fold). In some instances, butyrate caused a decrease in enzyme activity. There was no statistically significant correlation between the induction of Alkpase activity and that of other enzyme activities by sodium butyrate. Levels of aminopeptidase and DAP-IV activity were found to be dependent on cell density and increased 3- to 4-fold by the tenth day in most of the cell lines. Sodium butyrate altered the subcellular distribution pattern of the disaccharidases, causing a significant increase in activity associated with the soluble (cytoplasmic) fraction. Other enzymes such as Alkpase and DAP-IV continued to be predominantly associated with the membrane fraction in butyrate-treated cells. These data suggest that brush border membrane hydrolase activity and the effect of sodium butyrate may provide useful information regarding the differentiation of human colorectal cancer cells.

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine.

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.

Mucins secreted by cell lines derived from colorectal mucinous carcinoma and adenocarcinoma.

Mucinous (colloid) carcinoma and well- to moderately-differentiated adenocarcinoma of the colon differ in the pattern and the amount of mucin secretion and perhaps in their behaviour and clinical outcome. To ascertain why these differences exist and to elucidate the mechanisms of tumour progression, we examined two model human cell lines derived from colorectal mucinous carcinoma (C1a) and moderately differentiated adenocarcinoma (HM3) which show typical pathological and mucin staining patterns of the respective type of carcinomas to nude mouse tumour xenografts. Specifically, we sought to determine if there were quantitative and qualitative differences in mucin synthesis, in mucin gene expression and in biological properties between the two model cell lines. Northern blot analysis showed that MUC2 mRNA levels were significantly higher in C1a cells compared with HM3 cells, while those of MUC3, -5 and -6 mRNA were lower. C1a cells secreted approximately five times more radiolabelled apomucin and 1.5 times more glycosylated apomucin than HM3 cells. When the carbohydrate side-chain length of secreted mucins by these cell lines were examined by beta-elimination followed by P4 column chromatography, C1a mucins had mostly short carbohydrate side-chains, while HM3 cells had predominantly longer side-chains. Western blot analysis of the cell homogenate showed higher expression of MUC2 apomucin and mucin-associated carbohydrate antigens, such as T, Tn and sialyl Tn, with decreased sialyl Le(x) expression in C1a cells compared with HM3. Immunohistochemical analysis of 35 colorectal adenocarcinoma and 25 mucinous colorectal carcinoma tissues also demonstrated increased MUC2 apomucin, T, Tn and sialyl Tn antigens in the mucinous cancer specimens. Examination of the biological properties of these cell lines showed that C1a cells had significantly higher in vitro invasive activity in assays of invasion and collagenase activity and significantly lower E-selectin binding and liver colonisation activities in nude mice. These results indicate that colorectal mucinous carcinoma cells differ considerably from colorectal adenocarcinoma cells, both qualitatively and quantitatively, in the pattern of mucin gene expression and in the synthesis and secretion of mucin. In addition, biological studies showed that mucinous carcinoma cells have a greater degree of invasiveness, but less liver colonising activity. These results suggest that the biological and mucin characteristics of mucinous carcinoma cells contribute to extensive local invasion through tissue stroma as the predominant mechanism of tumour progression, while the biological and mucin characteristics of well- to moderately-differentiated colorectal adenocarcinoma contribute to progression via distant metastasis formation.

Protein digestion and absorption in man. Normal mechanisms and protein-energy malnutrition.

Protein is an essential nutrient normally assimilated in an efficient manner following the action of gastric, pancreatic and small intestinal enzymes. After hydrolysis, protein digestion products in the form of amino acids and small peptides undergo mucosal uptake by distinct transport mechanisms. Although gastric and pancreatic enzymes are important, the small intestine appears to be the critical rate-limiting tissue in this process. Impaired intake, assimilation or excessive enteric protein loss may occur with several diseases leading to protein-energy malnutrition. Although the clinical and laboratory features of this condition are nonspecific and wide ranging in spectrum, their presence may provide a clue to underlying disease and serve as an index of patient nutritional status. Disease of the exocrine pancreas or small intestine may cause significant protein-energy malnutrition which, in turn, can cause major structural and functional abnormalities in these tissues.

Chemoprevention of colon cancer carcinogenesis by balsalazide: inhibition of azoxymethane-induced aberrant crypt formation in the rat colon and intestinal tumor formation in the B6-Min/+ mouse.

Non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin have been shown to suppress colon carcinogenesis and in some cases reduce the size of colorectal polyps. Balsalazide disodium (BSZ) is a colon-specific prodrug of the salicylate, 5-aminosalicylic acid. The aim of the present study was to test the chemopreventive activity of BSZ in two established animal models of colon tumorigenesis, azoxymethane-induced aberrant crypt formation in the rat and intestinal tumor formation in the B6-Min/+ mouse. Aberrant crypt foci (ACF) were induced in Fischer 344 rats via 2 subcutaneous injections of azoxymethane (20 mg/kg). BSZ was supplied in the drinking water for 8 weeks and ACF quantitated. B6-Min/+ mice were treated from 55 days of age for 90 days and intestinal tumors scored for number, size and location. BSZ treatment of AOM-injected rats reduced ACF formation in a dose-dependent manner by 60% with the greatest effect observed on ACF with 4 or more crypts. In B6-Min/+ mice a dose-dependent reduction of intestinal tumor number was observed which reached 80% in the distal small intestine and colon. A preliminary mechanistic study in cultured human colon cancer cells showed that both BSZ and 5-ASA inhibited colon cancer cell proliferation in vitro. However, 5-ASA but not BSZ produced changes consistent with the induction of apoptosis. BSZ produces a dose-dependent chemopreventive effect on colon carcinogenesis. A possible mechanism is consistent with the inhibition of cellular proliferation and the induction of apoptosis.

Biological properties and expression of mucins in 5-fluorouracil resistant HT29 human colon cancer cells.

We have previously reported that HT29 human colon cancer cells selected by adaptation to 5-fluorouracil (5FU) (HT29-5FU cells) express increased levels of a major intestinal mucin MUC2 mRNA compared with parental HT29 cells. In this study, we examined in detail the changes in synthesis and secretion of mucin that occur in these cells and accompanying changes in the expression of cancer associated mucin related carbohydrate antigens and cell lineage associated biochemical markers. We further investigated their relationship to biological properties of cells. Northern blot analysis revealed a markedly increased level of MUC2 mRNA but no significant change in the mRNA levels of other mucins in HT29-5FU cells compared with parental HT29 cells. Labeling with radiolabeled precursors demonstrated increased synthesis and secretion of mucin glycoproteins by HT29-5FU cells. Immunoblot analysis showed a higher expression of mucin associated carbohydrate antigens such as T, Tn, sialyl Tn, sialyl Lea, sialyl Lex and non-O-acetylated sialic acid concomitant with significant increases in the expression of goblet cell lineage marker, MUC2 apomucin and a panepithelial cell marker, carcinoembryonic antigen. HT29-5FU cells showed significantly higher adhesion to E-selectin and to matrigel and in vitro invasive properties and significantly increased liver colonization capacity in nude mice following splenic vein injection. Nude mouse xenograft tumors produced by HT29-5FU cells showed a greater degree of differentiation, consisting of mucin secreting glands than those produced by parental HT29 cells. These results indicate that predominantly colonic type mucin, MUC2, has been selectively induced in HT29-5FU cells and that altered regulation of mucin genes associated with altered synthesis and secretion of mucin glycoproteins and the degree of differentiation in cancer cells may be responsible for the altered biological properties of these cells.

Phorbol 12-myristate 13-acetate induces alteration in mucin gene expression and biological properties of colon cancer cells.

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate diverse cellular responses through signal transduction pathways including the protein kinase C (PKC) pathway. In the present study, we sought to determine the effect of PMA on mucin gene expression and on the biological properties of a human colon cancer cell line, HM3. The cells were treated for 8 and 24 h with various concentrations of PMA and total RNA was extracted and Northern and slot blot analyses were carried out using MUC2, MUC3 and MUC5AC mucin cDNA probes to assess the steady state levels of mRNA. Spent media were collected and the level of cancer associated carbohydrate antigens (T, Tn, sialyl Tn, sialyl Lex, and sialyl Lea) and matrix-degrading metalloproteinase (MMPs) activity were examined. Trypsinized cells were used for assessing in vitro invasion, motility and adhesion to matrigel. Our results showed that PMA caused upregulation of steady state mRNA levels of MUC2, MUC3 and MUC5AC which was inhibited after treatment with protein synthesis inhibitors. Calphostin C, a highly specific inhibitor of protein kinase C significantly inhibited the PMA induced induction of mRNA levels of MUC2, MUC3, and MUC5AC. The levels of all cancer-associated mucin carbohydrate antigens examined in the media were increased by PMA treatment. PMA also caused an increase in MMPs activity and in in vitro invasion and motility properties, but did not affect adhesion of HM3 cells to matrigel. Thus, PMA caused a significant increase in the expression of all three mucin genes through signaling pathways involving protein kinase C and increased secretion of mucin associated carbohydrate antigens. These changes were associated with increases in MMP activity as well as by increases in the invasive and motility properties of HM3 colon cancer cells. These data suggest that protein kinase C signaling pathways may be involved in mucin gene regulation and in modulating the invasive and metastatic properties of colon cancer cells.

Gastric, pancreatic, and biliary secretion and the rate of gastric emptying after parietal cell vagotomy.

We compared the gastric, pancreatic, and biliary secretory responses to a liquid test meal and the rates of gastric emptying of liquid and solid test meals in six patients at least 1 year after parietal cell vagotomy with eight unoperated subjects, one with duodenal ulcer disease and seven normal control subjects. Parietal cell vagotomy decreased gastric acid secretion to one third of normal, but total trypsin and bile salt secretion during the first 150 postcibal minutes were normal. The liquid test meal emptied from the stomach faster after parietal cell vagotomy, the pattern of emptying being exponential in the vagotomy patients and linear in the normal subjects. The rate of gastric emptying of a liquid meal, although faster than normal, was less precipitous after parietal cell vagotomy than after truncal vagotomy plus drainage or subtotal gastrectomy, and trypsin and bile salt concentrations were not diluted to abnormal levels, as occurs after these other procedures. Furthermore, emptying and dispersion of solid food remained normal after parietal cell vagotomy. These findings probably explain, at least in part, the decreased incidence of postprandial dumping and diarrhea that accompanies parietal cell vagotomy compared with the other popular operations for duodenal ulcer.