Dietary regulation of ornithine transcarbamylase mRNA in liver and small intestine

In the rat, changes in dietary protein intake give rise to changes in the levels of ornithine transcarbamylase (OTC) in liver and small intestine--an increase in liver and decrease in small intestine. The changes in enzyme level are accompanied by similar changes in levels of specific mRNA. Thus in liver, there is an increase in the level of specific mRNA when protein intake is increased, whereas in small intestine there is a small decrease. Comparison of changes in specific mRNA with total poly-A-containing RNA showed that the change in OTC mRNA in liver paralleled the change in total RNA levels. In contrast, in small intestine the small decrease in OTC mRNA levels when protein intake was increased was in the face of an increase in the level of total mRNA. Whereas the level of OTC is 20-fold higher in liver than in small intestine, the mRNA level for the enzyme differs by only 2.5-fold.     

Radioimmunoassay studies of intestinal calcium-binding protein in the pig. I. Identification of intestinal calcium-binding protein in blood and response to a low calcium diet.

We have developed a radioimmunoassay for porcine intestinal calcium-binding protein (CaBP) and have used it to detect CaBP in pig plasma. Plasma CaBP is identical to intestinal CaBP on the basis of immunological activity, molecular size, and molecular charge properties. The plasma CaBP concentration was greater in the portal blood than in mixed venous blood, suggesting that blood CaBP originates in the gut. Two of four 15-week-old littermate pigs were placed on a low calcium diet (0.15% calcium, 0.65% phosphorus) and two on a control diet (0.65% calcium, 0.65% phosphorus). After 2 weeks, the entire small intestine was removed and divided into nine 1.8-m segments. CaBP was assayed in both plasma and intestinal mucosa. When the two pigs on a low calcium diet were compared with two control pigs, there was a general increase in immunoreactive CaBP in both plasma and intestinal mucosa. However, there was no increment in immunoreactive CaBP in the first 1.8-m segment of small intestine. Seventy-one percent of the increment in CaBP occurred distal to the first two segments. The largest fractional low calcium diet effect occurred in the ileum. The mean CaBP concentration for the total small intestine increased by a factor of 1.9. The plasma CaBP concentration increased by a factor of 2.6. In these pigs, plasma CaBP was a more reliable indicator of change in CaBP status than was the measurement in the proximal gut segment which contained the duodenum. The assay of CaBP in blood is convenient and may obviate the sampling errors inherent in intestinal biopsy.     

Distribution and abundance of nutrient transporter mRNA in the intestinal tract of the black bear, Ursus americanus

End products of digestion are absorbed by the body through the action of transporter proteins expressed on the apical membrane of intestinal epithelial cells. We investigated the mRNA abundance and distribution of a peptide transporter (PepT1), a glucose transporter (SGLT1), two amino acid transporters (NBAT and b(o,+)AT), and a digestive enzyme, aminopeptidase N (APN), in the intestinal tract of black bears (Ursus americanus). Intestinal total RNA was isolated from 10 bears and abundance of PepT1, SGLT1, NBAT, b(o,+)AT, and APN mRNA were determined by Northern blots. Abundance of PepT1 (P<0.05), APN (P<0.05), and SGLT1 (P<0.0001) changed quadratically from the proximal to distal intestine with abundance being greatest in the midregion. Abundance of b(o,+)AT mRNA increased linearly (P<0.05) from the proximal to distal intestine. The number of molecules of mRNA/ng of total RNA for each gene was determined using Real-Time PCR. PepT1 mRNA was present at 10-fold or greater levels than amino acid transporter mRNA in all segments of the intestine, suggesting that di- and tripeptides constitute a major form in which amino acids are absorbed in the black bear. The abundance of NBAT and b(o,+)AT mRNA was greater towards the distal intestine, suggesting a role in salvaging unabsorbed amino acids.     

The levels of lactase and of sucrase-isomaltase along the rabbit small intestine are regulated both at the mRNA level and post-translationally.

We determined along the small intestine of young and adult rabbits the activities of lactase (LPH) and sucrase (SI), the levels of their cognate mRNAs, and examined the in vitro biosynthesis of LPH and pro-SI. Lactase activity is low in the proximal 1/3 of the intestine, whereas the mRNA levels are high. However, the rates of biosynthesis of the LPH forms correlated well with the steady-state levels of LPH mRNA in all segments, indicating that factor(s) acting post-translationally produce a decline in brush border LPH in the proximal small intestine. These factor(s) are not involved in the processing of pro-LPH to mature LPH, since the relative amounts of the various forms of LPH are almost the same along the small intestine. Unexpectedly, we find that also for SI the ratio of activity to mRNA is low in proximal intestine. The biosynthesis of pro-SI correlates with the steady-state levels of its mRNA. Hence, the steady-state levels of LPH and SI along the small intestine are regulated both by mRNA levels and by posttranslational factor(s).     

Luminal and systemic signals trigger intestinal adaptation in the juvenile python

Juvenile pythons undergo large rapid upregulation of intestinal mass and intestinal transporter activities upon feeding. Because it is also easy to do surgery on pythons and to maintain them in the laboratory, we used a python model to examine signals and agents for intestinal adaptation. We surgically isolated the middle third of the small intestine from enteric continuity, leaving its mesenteric nerve and vascular supply intact. Intestinal continuity was restored by an end-to-end anastomosis between the proximal and distal thirds. Within 24 h of the snake's feeding, the reanastomosed proximal and distal segments (receiving luminal nutrients) had upregulated amino acid and glucose uptakes by up to 15-fold, had doubled intestinal mass, and thereby soon achieved total nutrient uptake capacities equal to those of the normal fed full-length intestine. At this time, however, the isolated middle segment, receiving no luminal nutrients, experienced no changes from the fasted state in either nutrient uptakes or in morphology. By 3 days postfeeding, the isolated middle segment had upregulated nutrient uptakes to the same levels as the reanastomosed proximal and distal segments, but it still lacked any appreciable morphological response. These contrasting results for the reanastomosed intestine and for the isolated middle segment suggest that luminal nutrients and/or pancreatic biliary secretions are the agents triggering rapid upregulation of transporters and of intestinal mass and that systemic nerve or hormonal signals later trigger transporter regulation but no trophic response.     

小鼠小肠四种消化酶活力的胎后发育模式

为明确晚成型小鼠胎后发育肠道消化酶活力的建立过程和发育模式,探讨其与适应性调节假说的关系,测定了从出生后至27日龄小鼠小肠前、中、后段的乳糖酶、蔗糖酶、麦芽糖酶和氨基肽酶的酶活力。结果发现单位组织酶活力方面,乳糖酶活力先增后降,小肠前段在9日龄而中后段在12日龄达到最高,至27日龄时仅中段有微弱的酶活力;蔗糖酶活力12日龄始出现,前段和后段自15日龄迅速升高,至18日龄达最高,但随后显著降低,而中段在15日龄后持续升高至21日龄达到最高,此后维持在较高水平;麦芽糖酶出生时已具有活力,但在15日龄前维持较低水平,此后迅速升高,前后段在18日龄,中段在21日龄达到峰值,此后下降;小肠前段的氨基肽酶活力出生后至27日龄持续下降,而后段和中段从出生到断乳前则持续升高,断乳后略有下降。除乳糖酶总酶活力先增后降,在15日龄达峰值外,其余3种酶的总酶活力均持续增加。在小肠不同位置4种酶活力的分布具有显著差异,且日龄对不同位置酶活力的影响趋势不同。总之,小鼠小肠4种消化酶的酶活力随时间的变化能够与其食物转变的消化需求相匹配,部分地支持适应性调节假说。     

Effects of diets containing soybean meal on trypsin mRNA expression and activity in Atlantic salmon (Salmo salar L)

Atlantic salmon develop subacute enteritis in the distal intestine (DI) when fed diets containing soybean meal (SBM) at high levels, a condition accompanied by increased trypsin activity in the DI intestinal content compared to fish fed conventional fishmeal (FM) based diets. To further investigate the responses of Atlantic salmon to dietary SBM, we measured trypsin activity in intestinal contents, quantified pancreatic trypsin mRNA expression, surveyed trypsin mRNA expression in selected tissues and characterized active forms of trypsin in the intestinal wall and brain. Enzyme measurements showed that trypsin activity in the intestinal content of SBM fed fish was lower in the proximal segments of the intestine, but higher in the DI compared to FM fed fish. The difference in enzyme activity was not reflected in a differential expression of pancreatic trypsin mRNA between fish fed the different diets (FM or SBM). Trypsin mRNA was expressed in 18 different tissues (esophagus, stomach, pancreas, pyloric tissue, midintestine, distal intestine, liver, head kidney, kidney, heart, spleen, thymus, brain, eye, gills, gonads, muscle and skin) but was most prominently expressed in tissues of the gastrointestinal (GI) tract and brain. We report for the first time an upregulation of trypsin-like activity in the DI wall using an in-gel trypsin activity assay, as well as modulated activity in the brain of fish fed SBM. The increased activity in the DI wall may contribute to disease severity and higher trypsin activity in the intestinal content.     

Effect of age and dietary calcium on intestinal calbindin D-9k expression in the rat

The capacity of rats and humans to adapt to low dietary Ca by increasing intestinal Ca absorption declines with age. The intestinal calbindin-D-9k protein (calbindin) is thought to play a role in the transcellular transport of Ca across the mammalian intestine. The purpose of these studies was to determine the effect of age and diet on the expression of calbindin at the protein and mRNA levels. Young (2 month) and adult (12 month) male F344 rats were placed on either a high Ca diet (1.2%) or a low Ca diet (0.02%) for four weeks. In the duodenum, the level of intestinal calbindin protein induced by a low Ca diet was 8-fold higher in young rats compared to adult rats. In the ileum, expression of calbindin protein was only about 10% that of the duodenum. In addition, the adult ileum showed the same decreased adaptation to a low Ca diet that was seen in the adult duodenum. In both the duodenum and the ileum, the changes in calbindin protein expression were highly correlated with calbindin mRNA expression and the correlations in each segment were quantitatively similar. In the duodenum, the changes in calbindin protein levels were strongly correlated with both Ca transport and Ca uptake. This quantitative correlation suggests a role for calbindin protein in the age-related decline in Ca absorption. In the ileum, the decreased adaptation to a low Ca diet may also be important given the long transit time through the distal intestine. The changes in both intestinal segments may contribute to the negative Ca balance seen in adult rats fed a low Ca diet.     

In vivo fractional P(i) absorption and NaPi-II mRNA expression in rainbow trout are upregulated by dietary P restriction

Mammalian type II sodium-phosphate cotransporter (NaPi-II) and inorganic phosphate uptake stimulator (PiUS) genes are upregulated by dietary phosphorus (P) restriction to increase intestinal and renal P transport, but little is known about NaPi-II and PiUS regulation in other vertebrates. We studied the 1). the tissue distribution and dietary regulation of NaPi-II, PiUS, and sodium-glucose cotransporter (SGLT1) mRNA and NaPi-II protein in juvenile rainbow trout (Oncorhynchus mykiss) and 2). effects of dietary P on intestinal Pi absorption in vivo. NaPi-II, PiUS, and SGLT1 mRNA were found in the proximal and distal intestine, pyloric ceca, and kidney. PiUS mRNA was also found in the heart, gill, blood, stomach, liver, skin, and muscle. Tissue distribution of NaPi-II protein correlated with that of NaPi-II mRNA except in gill ionocytes where NaPi-II antibodies recognized related epitopes. Chronic consumption of a low-P diet increased NaPi-II and PiUS but not SGLT1 mRNA abundance in the intestine and kidney. Unlike mammals, there was no detectable shift in tissue or cellular localization of NaPi-II protein in response to dietary P restriction. Regulation of NaPi and PiUS mRNA expression was observed only in fish grown under optimal aqueous oxygen concentrations. In vivo fractional absorption of Pi by the intestine decreased in fish fed high-P diets. Decreases in absorption were less pronounced in fish previously fed low-P diets, suggesting that diet history modulates acute regulation of P absorption. Regulation of dietary Pi absorption in vivo may involve a specific change in intestinal NaPi-II and PiUS gene expression.     

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.     

Effects of dietary glucose and sodium chloride on intestinal glucose absorption of common carp (Cyprinus carpio L.)

The co-transport of sodium and glucose is the first step for intestinal glucose absorption. Dietary glucose and sodium chloride (NaCl) may facilitate this physiological process in common carp (Cyprinus carpio L.). To test this hypothesis, we first investigated the feeding rhythm of intestinal glucose absorption. Carps were fed to satiety once a day (09:00 a.m.) for 1 month. Intestinal samples were collected at 01:00, 05:00, 09:00, 13:00, 17:00 and 21:00. Result showed that food intake greatly enhanced sodium/glucose cotransporter 1 (SGLT1) and glucose transporter type 2 (GLUT2) expressions, and improved glucose absorption, with highest levels at 09:00 a.m.. Then we designed iso-nitrogenous and iso-energetic diets with graded levels of glucose (10%, 20%, 30%, 40% and 50%) and NaCl (0%, 1%, 3% and 5%), and submitted to feeding trial for 10 weeks. The expressions of SGLT1 and GLUT2, brush border membrane vesicles (BBMVs) glucose transport and intestinal villus height were determined after the feeding trial. Increasing levels of dietary glucose and NaCl up-regulated mRNA and protein levels of SGLT1 and GLUT2, enhanced BBMVs glucose transport in the proximal, mid and distal intestine. As for histological adaptive response, however, high-glucose diet prolonged while high-NaCl diet shrank intestinal villus height. Furthermore, we also found that higher mRNA levels of SGLT1 and GLUT2, higher glucose transport capacity of BBMVs, and higher intestinal villus were detected in the proximal and mid intestine, compared to the distal part. Taken together, our study indicated that intestinal glucose absorption in carp was primarily occurred in the proximal and mid intestine, and increasing levels of dietary glucose and NaCl enhanced intestinal glucose absorption in carp.     

Biosynthesis of apolipoprotein C-III in rat liver and small intestinal mucosa

We have examined the biosynthesis of rat apolipoprotein C-III in the small intestine and liver. The primary translation product of its mRNA was recovered from wheat germ and ascites cell-free systems. Comparison of its NH2-terminal sequence with the NH2 terminus of plasma high density lipoprotein-associated apolipoprotein C-III showed that apo-C-III was initially synthesized as a preprotein with a 20 amino acid long NH2-terminal extension: Met-X-X-X-Met-Leu-Leu-X-X-Ala-Leu-X-Ala-Leu-Leu-Ala-X-Ala-X-Ala. Co-translational cleavage of the cell-free translation product by signal peptidase generated a polypeptide with the same NH2 terminus as the mature protein (X-Glu-X-Glu-Gly-Ser-Leu-Leu-Leu-Gly-Ser-Met). Therefore, this apolipoprotein does not undergo post-translational proteolytic processing like two other high density lipoprotein-affiliated proteins, proapo-A-I and proapo-A-II. The mRNA encoding apolipoprotein C-III comprises 0.4% of the translatable RNA species in adult rat liver and 0.14% of the translatable RNA species in small intestinal epithelium. Acute fat feeding with a triglyceride meal resulted in a 2-fold increase in intestinal preapo-C-III mRNA accumulation but no change in the levels of preproapo-A-I mRNA. Thus, the acute response of the apo-A-I and C-III genes to triacylglycerol absorption differs.     

Intestinal absorption of hexoses in rats infected with Nippostrongylus brasiliensis

The net absorption and accumulation of d-galactose and d-glucose by the small intestine of rats infected with N. brasiliensis were studied in vivo and in vitro. There was no change from control levels in the rate of galactose transfer in vivo by the entire intestine 10 days after infection but fluid transfer was significantly lower at this time. Mucosal galactose transfer in vitro by the entire intestine or by each one-third of the intestine did not change significantly during infection but 10 days after infection mucosal glucose transfer was significantly lower in the infected proximal one-third of the intestine and significantly greater in the distal one-third than in the comparable segments in controls; mucosal glucose transfer by the entire intestine was not affected by infection. Serosal transfer of both hexoses by the proximal two-thirds of the intestine and by the entire intestine was significantly reduced 10 days after infection. Between 10 and 18 days after infection the rate of serosal galactose transfer in vitro was significantly lower than control levels. The difference in response of mucosal and serosal hexose transfer rates to infection appears to be due, in part, to an increase in intestinal glucose metabolism or increased tissue retention of galactose during infection. Mucosal fluid transfer in vitro by the entire intestine was not significantly different from control levels at 10 days of infection when either hexose was used, although there was a significant reduction in the jejunal segment when glucose was used. Mucosal fluid transfer by the entire intestine in the presence of galactose was significantly greater during the rejection phase of the parasite population than in controls.     

Expression of the peptide transporter hPepT1 in human colon: a potential route for colonic protein nitrogen and drug absorption

Substrates of the proton-coupled peptide transporter, hPepT1, include dietary di- and tripeptides plus therapeutically important drugs such as the beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Expression and function of hPepT1 in the small bowel is well established. We have compared levels of hPepT1 mRNA expression in regions of human gut by RT-PCR methods and examined the expression of hPepT1 in normal human colon using an anti-hPepT1 antipeptide antibody. hPepT1 mRNA was expressed in the large intestine, although at lower levels than in the small intestine. Quantitatively, expression in ileum was 4.6-fold greater than in sigmoid colon. Immunoreactive hPepT1 was detected in human colon at lower levels than in ileum. The pattern of expression differed between the two tissues: whilst expression in the ileum was localised to the apical enterocyte membrane along the length of the crypt-villus axis, expression in the colonocyte was detected at the apical membrane towards the luminal surface but predominantly at the basal membrane towards the base of the crypt. We conclude that distal regions of the bowel express hPepT1, which may provide a mechanism for colonic protein-nitrogen absorption and for absorption of therapeutically important peptidomimetic drugs.