Lower Villus Research Articles

Localization of acyl-coenzyme A: cholesterol acyltransferase in villus and crypt cells of chick intestine

Endogenous cholesterol esterification by acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26) was studied in isolated enterocytes obtained from chick duodenal, jejunal, and ileal villi and crypts, using [14C]oleoyl-CoA as substrate. The maximal specific activity in each cell fraction was found in chick jejunum, followed by duodenum and ileum. Jejunal upper and mid villi showed higher specific activities than lower villi and crypts. Epithelial cells isolated from chick intestine also incorporated oleoyl-CoA into different lipids using the endogenous substrates. Upper and mid villus cells showed the maximal incorporation of oleoyl-CoA into triglycerides in duodenum and jejunum. Levels of oleoyl-CoA incorporation into phospholipids were higher than those found in the synthesis of triglycerides or cholesterol esters, whatever may be the cell fraction considered. Upper villus cells also showed the highest specific activity in the incorporation of oleoyl-CoA into phospholipids. The acyl-CoA hydrolase specific activity was practically similar in all the cell fractions obtained from chick duodenum, jejunum, and ileum.

Dietary CHO and stimulation of carbohydrases along villus column of fasted rat jejunum

Adult rats when fed a high carbohydrate diet of 70% sucrose or glucose for 24 h following a 4-day fast showed increased concentrations of intestinal sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) but not lactase-phlorizin hydrolase (EC 3.2.1.23, EC 3.2.1.62). The concentration increases of these enzymes were accompanied by corresponding acceleration of their synthesis rates. Contrary to earlier studies by others, suggesting that upper villus cells in the fasted intestine are unresponsive to stimulation of sucrase activity by refeeding a high-sucrose diet, the concentration increases of both sucrase-isomaltase and maltase-glucoamylase were seen to occur in cells all along the length of the villus column. The earlier studies differed from the present study by basing enzyme assays relative to protein rather than the DNA content of villus cell fractions. We have shown that villus cells increase their protein content severalfold while migrating to villus tip, providing the basis for the difference between earlier and the present findings. Further evidence that stimulation of sucrase-isomaltase and maltase-glucoamylase by high carbohydrate is not restricted to the crypt and lower villus region was obtained by the finding that their synthesis rates appeared to be equally stimulated along the length of the villus column.

Using immunological criteria to predict utilization of soybean proteins by the early-weaned pig (1989)

Two trials were conducted to determine the suitability of soybean products for baby pigs. For trial 1, 40 weanling pigs were orally infused with .01 lb/pig/d of either dried skim milk (control), soybean meal (48% CP), soy protein concentrate, extruded soy protein concentrate, or soy protein isolate from d 7 to 12 of age, weaned at 21 d of age, and fed a diet containing the corresponding protein source until d 7 postweaning. Sows were fed a corn-corn gluten meal-based diet supplemented with lysine and tryptophan to avoid exposure of pigs to soybean proteins. All pigs were sacrificed at 28 d of age. In Trial 2, 48 pigs were utilized, with preweaning treatments identical to those in Trial 1 except the soy protein isolate was not used as a treatment. They were fed a diet containing the same protein source for 2 wk postweaning, then fed a common diet with 4% soybean oil and 1.25% lysine for 3 wk. Growth performance was measured. Results indicated that pigs fed diets containing soybean meal had lower villus height and rate of gain than pigs on any other treatments. There were no differences in villus height and crypt depth among soy protein concentrate, extruded soy protein concentrate, and soy protein isolate. In the growth trial, pigs fed the diet containing extruded soy protein concentrate had the highest ADG compared to other soybean products tested. Decreased villus height and increased serum anti-soy IgG titers, coinciding with inferior performance and presence of residual antigenic protein in the digestive tract of baby pigs fed soybean meal, indicate that conventionally processed, commercial soybean meal retain antigens that cause immunological changes in early-weaned pigs.; Swine Day, Manhattan, KS, November 16, 1989

Golgi apparatus and its structural differentiation in duodenal epithelial cells.

Using cytochemical techniques, structural details of the Golgi apparatus were investigated under conventional and high voltage electron microscopes. Mouse duodenal absorptive epithelial cells in the crypt-base show cellular maturation during cell migration toward the tip of villus. The Golgi apparatus and its related structures also manifest several remarkable differences between immature cells located in lower part of the crypt and the mature cells in lower villus or midvillus. In immature cells in the crypt, the Golgi apparatus was well organized; simple stacking of cisternae and sparsely distributed rough ER often show face-to-face attachments not only to the trans-most cisterna of the Golgi stack, but also to the cis-most. In mature cells located at villus-base or mid-villus, the Golgi stack shows considerable development: a wavy folding to form a large highly-complex network. In the trans-side, at least three kinds of structures can be distinguished: lamellar cisterna, large-sized lipid granules (0.3-1.0μm in diameter) and trans-tubular network. The lamellar cisterna shows extraordinary independence from other Golgi stack, and it tends to detach or “peel off” from the Golgi stack. These three structures are related by structural transitions. The latter two components appeared to derive from the lamellar cisterna. In the cis-side, besides the meshed cisterna being characteristic to the cis-most cisterna, small-sized lipid granules (50nm in diameter) were often observed in the cis-most cisterna. Furthermore, cis-most cisternae have direct connections to tubular structures which extended from the apical cytoplasm. This structure is different from the trans-tubular network and contain small lipid granules identical to those observed in cis-most cisterna. From these observations, we postulate that substantial transport through Golgi apparatus might be promoted by cisterna progression from cis to trans side.

The role of enterocyte cholesterol metabolism in intestinal cell growth and differentiation.

Cholesterol is an essential constituent of all mammalian cell membranes, and its availability is therefore a prerequisite for cellular growth and other functions. To define further the role of cholesterol metabolism in the intestine both in vitro and in vivo, studies were performed. Several lines of evidence based on these studies suggest that the main purpose of local cholesterol synthesis in the gut is the support of rapid enterocyte proliferation: 1) growth was inhibited during pharmacologic suppression of cholesterol synthesis in intestinal organ or cell culture; 2) the endocrine regulation of intestinal growth was in most but not all instances accompanied by appropriate changes in cholesterol synthesis; 3) most of cholesterol synthesis and lipoprotein uptake was localized predominantly in the crypt and lower villus region; and 4) very little of the sterol synthesized by the intestinal mucosa was exported into lymph but seems rather to be incorporated into cell membranes.

Expression of Developmentally Regulated Crypt Cell Antigens in Human and Rat Intestinal Tumors<xref ref-type="fn" rid="FN1">2</xref>

The expression of 5 antigens specific for adult intestinal crypt cells and defined by monoclonal antibodies prepared to surface membrane components of the human colon tumor cell line CaCo-2 was studied during fetal and postnatal development of Sprague-Dawley rat small and large intestines. In the small intestine, all epithelial cells were stained during fetal life; antigen distribution became restricted to the crypt and lower villus cells in suckling animals and to the crypt cells after weaning. In the colon, these antigens could be detected only during a short period of development, comprising the last 3-4 days of fetal life and the first 8-10 days after birth. Expression of the antigens defined by this group of antibodies was investigated in rat intestinal tumors induced by 1,2-dimethylhydrazine (CAS: 540-73-8) and in normal and diseased human colons. In rats, these antigens were detected in all poorly and moderately differentiated adenocarcinomas of the small and large intestines. In most specimens, antigen distribution was not uniform; intensely stained areas were surrounded by completely negative tumor regions. Antigen expression was less intense in well-differentiated tumors, and about half of the tumors were negative for antigen expression. A similar pattern of expression of these antigens was observed in all human colonic adenocarcinomas examined; all samples of normal colon, benign polyps, and inflammatory bowel diseases examined were negative. These results suggest that this group of monoclonal antibodies recognizes oncofetal rat antigens expressed in chemically induced rat intestinal tumors and human colonic adenocarcinomas.

Fetal characteristics of small intestinal crypt cells.

Nine monoclonal antibodies were prepared against luminal membranes purified from rat intestinal cells at day 19 of gestation, and seven of them were found to define antigens common to adult crypt cells and fetal or embryonic intestinal epithelial cells. The FBB 2/29 antigen was first detected over the entire intestinal epithelial population at days 14-15 of gestation, a period of development characterized by formation of a stratified intestinal epithelium and differentiation of the surrounding mesenchyme. This antigen, identified as a set of high molecular mass proteins, became restricted to the crypt and lower villus cells after birth and was exclusively expressed by the crypt cells in adult intestine. It also was found to be expressed by the epithelial cells of the distal tubuli in the kidney of adult rats and by cultured human tumor colonic cells. The FBB 1/54/1, FBB 3/46, and FBB 3/78/9 antibodies stained only the epithelial cells present at the base of the villi in fetal intestine, starting at days 20-21 of gestation (about 1-2 days before birth), and stained the crypt and lower villus cells in newborn and adult intestine; these antigens may be regarded as specific markers for the developing crypt cells in fetal intestine shortly before birth. The FBB 1/20 and FBB 4/2 antigens were first detected on the fetal intestinal cells at day 18 of gestation; they were located over the entire epithelium in newborn rats and became restricted to the crypts after weaning. The FBB 2/28 antigen was expressed by the entire intestinal epithelium at all stages of development, starting from days 18-19 of gestation in the fetus. Two antibodies, FBB 3/4 and FBB 3/24, were found to be specific for lactase. These results have demonstrated the expression of cell- and tissue-specific components in rat intestine during early embryonic development and revealed a marked similarity in surface membrane antigens between fetal intestinal epithelial cells and adult crypt cells.

Glycoconjugate distribution and mobility on apical membranes of absorptive cells of suckling rat ileum in vivo.

The luminal membrane of ileal absorptive cells in suckling rats includes two domains: microvillar membranes and deep invaginations between microvilli. We examined the fates of foreign macromolecules that bind to anionic or saccharide sites on these domains after infusion into ligated loops in vivo. Cationized ferritin (CF) and ferritin-RCAI (beta-galactosyl) binding sites were distributed over the entire apical membrane. Ligands bound to apical invaginations were rapidly endocytosed, but ligands on microvilli were not. After CF binding, anionic sites on microvilli were mobile in the plane of the membrane and formed CF clusters at the tip and base of each microvillus. RCAI binding sites did not cluster. Wheat germ agglutinin (WGA, sialic acid) labeling was restricted to microvillus tips of mature cells but was dispersed over the microvillar surfaces of lower villus cells. Ferritin conjugates of Concanavalia ensiformis (Con A), Ulex europaeus agglutinin (UEA), and Dolichos biflorus agglutinin (DBA) did not bind to cell surfaces in vivo. Aldehyde fixation dramatically altered lectin binding patterns, resulting in unmasking and labeling of Con A, WGA, and DBA binding sites that were unavailable in vivo.

Specific binding of high density lipoprotein (HDL3) is not related to sterol synthesis in rat intestinal mucosa.

There is good evidence that high density lipoprotein (HDL) is involved in the flux of cholesterol into the cells of some organs and out of the cells of other tissues. Because we have previously found that HDL is bound specifically by mucosal cells of the small intestine, we have examined the possibility that this was associated with regulation of cholesterol flux. We have, therefore, compared the specific binding of 125I-labeled HDL3 with cholesterol synthesis in mucosal cells obtained from rats that had been treated to alter intestinal cholesterol metabolism. The rate of sterol synthesis measured in tissue slices, by the incorporation of [3H]water into sterols, was altered up to fivefold by treatment with cholestyramine (to induce bile salt loss), by surformer treatment (to reduce absorption of cholesterol), and by biliary diversion. Yet the capacity of mucosal cells to bind, internalize, and degrade 125I-labeled HDL3 was unchanged. Cholesterol feeding influenced neither the interaction of 125I-labeled HDL3 with cells nor the rate of sterol synthesis. Furthermore, the interactions of 125I-labeled HDL3 with mucosal cells isolated from the proximal and distal halves of the intestine or between the upper and lower villus cells were similar, despite differences in sterol synthesis. These data suggest that, in rat intestine, the specific binding of HDL is not related to sterol synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Crypt cell development in newborn rat small intestine.

Three monoclonal antibodies were prepared against luminal membranes from small intestinal cells of 3-d-old rats (YBB 1/27, YBB 3/10) and crypt cell membranes from adult rats (CC 4/80). The antibodies were shown to define specific stages of development of the intestinal crypt cells. The YBB 1/27 antigen was first detected at the luminal membrane of the epithelial cells in fetal intestine at day 20 of gestation; it was confined to the crypt cells and lower villus cells between 1 and 20-22 d after birth, and could not be detected in any region of the intestine in older animals. The YBB 3/10 antigen, identified as a set of high Mr proteins, was localized over the entire surface membrane of fetal intestinal cells and of crypt and villus cells after birth; after weaning (20-22 d after birth) it gradually disappeared from the villus cells and became confined to the region of the crypts. The CC 4/80 antigen, identified as a protein (or a set of related proteins) of molecular mass 28-34 kD, was shown to appear in the crypt cells 10-14 d after birth. Its distribution changed after weaning, when it disappeared from the crypts, and was localized in the absorptive lower villus cells. This change in pattern could, in part, be prematurely elicited by cortisone injection in younger animals. These results have demonstrated the presence of specific surface membrane components on the intestinal crypt cells, and suggested that fetal antigens may be retained in these cells after birth.

655 TIME AND DOSE DEPENDENT EFFECT OF SUCROSE AND LACTOSE INTAKE ON SUCRASE AND LACTASE ACTIVITIES IN ADULT RAT JEJUNUM

To characterize mechanisms leading to a dietary evoked increase of sucrase (Sa) and lactase (La) activity by dietary intake of disaccharides [sucrose (S) or lactose (L)], two-month-old rats were fed for 7 days a low carbohydrate diet (LC) [BBA 676:108], then for the next 3 to 72 hours, they were force fed a S or L diet (20 or 40 cal%). Activity of La and Sa increased after 3 hours, but only Sa continued to increase for the next 48 hours. The effect of variation of carbohydrate intake on Sa and La after 12 hours is summarized below. Whereas Sa increased with greater intake of S and L, La did not. Determination of changes of enzyme activity along the height of the villus-crypt columns showed that the increase of Sa began at the lower villus, but the increase of La occurred along the entire villus. Conclusion. Differences in response of Sa and La to S arid L intake (time, dose, locus) suggest that the increase of both disaccharidases evoked by dietary intake of S or L is the result of different mechanisms.

The temporal distribution of the 1 alpha,25-dihydroxycholecalciferol receptor in the rat jejunal villus.

The distribution of the 1 alpha,25-dihydroxycholecalciferol receptor was studied in enterocytes isolated from the upper, mid and lower villus and crypt cells of the jejunum of normal and rachitic rats. In all cell fractions a high-affinity receptor (KD congruent to 0.07 nmol/l) with a sedimentation coefficient of 3.5S was demonstrated. In normal rats there was a 60% reduction in receptor numbers in crypt cells compared with the mid and upper villous cells. Vitamin D deficiency led to a reduction in receptor numbers in all cell fractions (45% upper villus, 78% crypt cells). The data are compatible with the concept of calcium absorption occurring in the differentiated villous cells and also account for the reduction in absorption in rachitic animals.

Synthesis and coenzyme A-dependent esterification of cholesterol in rat intestinal epithelium. Differences in cellular localization and mechanisms of regulation.

The current studies were undertaken to characterize the localization and regulation of cholesterol synthesis and acyl-CoA:cholesterol acyltransferase activity in rat intestinal crypt and villus cells. Both parameters were determined in groups of animals with widely varying sterol fluxes across the intestinal mucosa. In animals on control diet the rates of cholesterol synthesis, measured by the incorporation of [3H]water per mg of protein, were similar along the villus/crypt axis in the jejunum, whereas in the ileum, villus cells were significantly more active than crypt cells. In both areas, however, the majority of total synthetic activity was found in cells from the crypts and lower villi. In contrast, the highest specific and total acyl-CoA:cholesterol acyltransferase activity was recovered in the villus cells of the jejunum and ileum. Dietary cholesterol did not affect sterol synthesis in any of the cell fractions but increased acyl-CoA:cholesterol acyltransferase activity approximately 2-fold in jejunal cell fractions. Inhibition of cholesterol absorption or sequestration of intestinal bile acids stimulated sterol synthetic activity up to 7-fold, and this occurred mainly in the lower villus and crypt cells in both jejunum and ileum. An increased demand for lipoprotein cholesterol, generated by triglyceride feeding, similarly was associated with enhanced synthetic rates. However, unlike cholesterol feeding, these manipulations did not increase acyl-CoA:cholesterol acyltransferase activity in any of the villus cell fractions. These studies suggest, therefore, that the intracellular pools of cholesterol that regulate the rate of cholesterol synthesis and the rate of cholesterol esterification are functionally distinct.

Absolute rates of cholesterol synthesis in rat intestine in vitro and in vivo: a comparison of different substrates in slices and isolated cells

In an effort to localize small intestinal sterol synthesis both along the length of the intestine and along the villus-crypt axis, we defined the optimal conditions to measure absolute rates of digitonin-precipitable sterol (DPS) synthesis in whole intestinal slices and in isolated epithelial cells. When [1-(14)C]acetate and [1-(14)C]octanoate were compared in midgut slices, [1-(14)C]octanoate was preferentially metabolized to CO(2) and DPS. Because of its rapid intramitochondrial degradation, [1-(14)C]octanoate effectively swamped-out other sources of acetyl CoA (C(2)) and achieved rates of C(2) flux into DPS virtually identical to the absolute rates determined with [(3)H]water. On the other hand, unlabeled acetate decreased the apparent C(2) flux from [1-(14)C]octanoate into DPS, but not into CO(2), in a dose-dependent fashion up to 8 mM. Under comparable incubation conditions, intestinal epithelial cells isolated by the EDTA chelation technique differed from slices in several respects. First, DPS synthesis from the various substrates was proportional to time only for 30 min compared to 90 min in slices. Second, the preferred (14)C-labeled substrate for DPS and CO(2) synthesis was acetate rather than octanoate. Third, neither of the (14)C-labeled substrates achieved the rates of synthesis found with [(3)H]water. Fourth, sterol synthesis from any of the substrates was essentially zero in the absence of glucose in the incubation medium. When the optimal rates of sterol synthesis in vitro were estimated using [(3)H]water in villus and crypt cell fractions of the jejunum and ileum, all ileal fractions were more active. However, ileal villus cells exceeded the jejunal villus cells by 5.2-fold in their capacity to incorporate [(3)H]water into DPS, whereas the crypt cell fractions differed by only 1.4-fold. The majority of the sterol synthetic capacity resided in the lower villus region in both the proximal and distal intestine and the crypts accounted for about 30% and 14%, respectively, of the total found in the jejunum and ileum. A similar distribution pattern along both the vertical and horizontal axes of the intestine was found after [(3)H]water administration in vivo, although under these conditions the proportion recovered in the crypts increased to 38% and 31%, respectively, of the total [(3)H]DPS found in the jejunum and ileum. These studies demonstrate that [(3)H]water yields optimal rates of sterol synthesis both in intestinal slices and in isolated epithelial cells and is incorporated into DPS mainly in cells of the lower villus and crypt region both in vitro and in vivo.-Stange, E. F., and J. M. Dietschy. Absolute rates of cholesterol synthesis in rat intestine in vitro and in vivo: a comparison of different substrates in slices and isolated cells.

Thyroxin‐Evoked Increase of Sucrase Activity in Lower Villus in the Jejunum of Adrenalectomized Suckling Rats

SummaryIntestinal sucrase activity, absent in the suckling rat, is evoked precociously by thyroid hormones. Since enterocytes migrate from crypt to villus tip, the question arises: at which level of the villus–crypt columns do the enterocytes respond with an increase of sucrase activity to thyroxin (T4)? Suckling rats (11 days old) were injected daily, subcutaneously, with T4 (2 μg/g BW/day). They were sacrificed 1, 2, and 3 days later. Sucrase activity was determined in homogenates of the entire jejunal wall and in serial homogenates of the villus–crypt columns using cryostat sectioning. Maximal increase of sucrase activity was seen after 3 days in the lower villus. Experiments were also done using rats adrenalectomized on day 10 to exclude the effect via precocious maturation of adrenal cortex. The same results were obtained. In conclusion, T4‐evoked increase of sucrase activity occurs first in enterocytes that are nearest the villus–crypt junction.

The possible role of calcium-binding protein induced by 1 alpha,25-dihydroxyvitamin D3 in the intestinal calcium transport mechanism.

The relationship between the appearance of vitamin D-dependent calcium-binding protein (CaBP) and calcium absorption was studied in sequentially isolated duodenal mucosal preparations from vitamin D-deficient chicks and those supplemented with vitamin D3 or 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. The duodenal calcium absorption activity was significantly increased 6 h after a single iv injection of 0.625 microgram of 1 alpha,25-(OH)2D3, attained a maximum at 12 h, and declined gradually thereafter. On the other hand, only small amounts of CaBP appeared in the crypt and lower villus regions 6 h after 1 alpha,25-(OH)2D3 administration. At 12 h, the CaBP was found in the entire villus, but its content was still much higher in the crypt and lower villus. At 24 h and 48 h, the distribution of CaBP showed the opposite gradient, higher in the villus and lower in the crypt. At 72 h, CaBP was found only in the upper villus. The life time of duodenal mucosal cells was calculated to be 108 h as indicated by the cells labeled with [3H]thymidine. Thus, the movement of CaBP from the crypt to the villus tip was considered to be much faster than the cell migration, suggesting that both crypt and villus cells are capable of producing CaBP. Daily administration of vitamin D3 or 1 alpha,25-(OH)2D3 for 2 weeks resulted in a marked increase in CaBP levels mainly in the mid- and upper villus regions. The higher the intestinal calcium transport activity was, the higher the duodenal CaBP content. These results suggest that CaBP is not necessary in initiating intestinal calcium transport, but it plays an important role in maintaining the enhanced transport mechanism.

Characterization of isolated epithelial cells from rat small intestine.

Intestinal mucosal cells from the rat have been isolated by a new technique involving intravascular perfusion of an intestinal segment with collagenase. Detached cells were flushed from the intestinal lumen with a second perfusion circuit containing an oxygenated buffered solution with 1% bovine serum. Sequential collection of cells at intervals during the period of perfusion revealed that villus-tip cells are recovered first (after 15 min of collagenase perfusion), followed by midvillus (after 25 min) and lower villus cells (after 35 min). The isolated cells were judged intact and viable by the criteria of trypan blue dye exclusion, ultrastructural appearance, and metabolic activity. They were characterized as villus-tip, midvillus, and lower villus-crypt cells by their alkaline phosphatase and sucrase activity, glycoprotein formation, and [3H]thymidine incorporation. Microsomal monooxygenase activity was four to five times greater in villus-tip than in lower villus cells, whereas heme oxygenase exhibited a reverse gradient. The isolated cells synthesized heme and bilirubin under cell culture conditions.

Effect of sucrose refeeding on disaccharidase and aminopeptidase activities of intestinal villus and crypt cells in adult rats. Evidence for a sucrose-dependent induction of sucrase in the crypt cells.

In order to gain more insight into the adaptative mechanism of intestinal enzymes to dietary factors in rats, modifications in the activities of disaccharidases and aminopeptidase were measured after refeeding of a 70% solution of sucrose for 15 h following a 2-day fast. Mature epithelial cells from the villus and immature cells from the crypt were isolated after sequential removal of the cells along the villus-crypt axis. Synthesis of brush border disaccharidases was determined by measuring [3H]valine incorporation into proteins. 1. In the whole mucosa, a highly significant increase in sucrase and maltase activities and a significant drop in aminopeptidase activity was observed in the brush border membranes after sucrose refeeding. 2. Stimulation of sucrase and maltase activities in sucrose refed rats was produced mainly in the immature cells of the crypt and lower villus compartment. 3. After separation of the brush border proteins by SDS gel electrophoresis from villus and crypt cells of sucrose refed rats, major incorporation of the radioactive precursor occured in the protein bands corresponding to sucrase and maltase activities of the lower villus and crypt cell brush borders. These findings demonstrate that sucrase stimulation by sucrose occurs mainly in the immature epithelial cells and that the substrate induces de novo synthesis of sucrase molecules.

Site of substrate stimulation of jejunal sucrase in the rat.

To identify the site of stimulation of sucrase by a sucrose diet, changes in sucrase-specific activity of jejunal mucosa were studied after introduction of sucrose diet to carbohydrate-deprived rats. Results were correlated with simultaneous changes in villus gradients of sucrase-specific activity. Simultaneous with the introduction of sucrose diet, [(3)H]thymidine (100 muCi) was administered intravenously, and rates of cell migration measured during adaptation to the new diet. After a 72-h fast, rats fed sucrose diet for 6, 12, or 18 h showed no change in sucrase-specific activity in either whole mucosa or villus gradients. However, within 18-24 h after starting a sucrose diet, there was a marked rise in whole mucosal sucrase-specific activity above fasting values (99 +/- 14 vs. 38 +/- 4 muM glucose/min per g protein, P < 0.001) in association with the development of a region of increased activity at the lower villus (154 +/- 22 vs. 60 +/- 9 muM glucose/min per g protein, P < 0.02, but with no change in villus tip activity (56 +/- 5 vs. 46 +/- 8 muM glucose/min per g protein). Similar changes were seen in animals fed 24 h of sucrose diet after a 72-h carbohydratefree diet. Fasted animals fed sucrose diet for 36 h had increased sucrase-specific activity at the villus tip (144 +/- 11 muM glucose/min per g protein) as well as at the lower villus region, and this pattern persisted at 1 wk of sucrose diet. Maximal activity patterns for isomaltase and maltase paralleled those for sucrase, but the villus gradients for lactase were unaffected by sucrose diet. The region of maximal sucrase-specific activity always coincided with or followed the leading edge of radioactivity as determined by liquid scintillation counting. Therefore, sucrose-mediated changes in sucrase activity of the jejunal mucosa in the rat appear to be initiated at the level of the crypt epithelial cell and are expressed after a latent period of 18-24 h during which these cells mature and migrate toward the villus tip.

Distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase and alkaline phosphatase activities in isolated ileal epithelial cells of fed, fasted, cholestyramine-fed, and 4-aminopyrazolo[3,4-d]pyrimidine-treated rats.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, E.C. 1.1.1.34), the major rate-limiting enzyme of the sterol biosynthetic pathway, was studied in ileal epithelial cells isolated in a villus-to-crypt gradient according to Weiser (Weiser, M. M. 1973. J. Biol. Chem, 248:2536-2541). Alkaline phosphatase (E.C. 3.1.3.1) served as a marker for the mature villus cells. Protease effects on activity determinations were negligible. The intracellular location of HMG-CoA reductase could not be precisely determined. The activity of ileal reductase was predominantly associated with the less differentiated lower villus and crypt cells, while the reverse gradient occurred with alkaline phosphatase. This distribution of enzymes persisted in both fed and fasted rats injected with control saline-phosphate, although fasting decreased total reductase units in the ileum by 86% in 72 hr. Treatment with cholestyramine and with 4-aminopyrazolo[3,4-d]pyrimidine (APP) enhanced reductase activity in ileal cells. The percent stimulation in both cases was higher in the upper villus cells than in the crypt cells, leading to abolition of the gradient in enzyme activity. However, APP treatment caused a 98% loss in total alkaline phosphatase units and a 55% loss in total epithelial cell protein in 72 hr. Thus, there was no increase in total reductase units. These data show that APP affects ileal cell metabolism directly. Furthermore, it appears that the regulation of sterol synthesis in the intestinal mucosa, via HMG-CoA reductase, involves a complex interplay of the effects exerted by the level of alimentation, the enterohepatic circulation of bile, and the levels of plasma lipoproteins.