Changing genes; losing lactase.
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Gene Expression Regulation, Enzymologic↗
Biomedical subjects
R K Montgomery
Publications and source records attributed to R K Montgomery.
Development of the human gastrointestinal tract: twenty years of progress.
A combination of approaches has begun to elucidate the mechanisms of gastrointestinal development. This review describes progress over the last 20 years in understanding human gastrointestinal development, including data from both human and experimental animal studies that address molecular mechanisms. Rapid progress is being made in the identification of genes regulating gastrointestinal development. Genes directing initial formation of the endoderm as well as organ-specific patterning are beginning to be identified. Signaling pathways regulating the overall right-left asymmetry of the gastrointestinal tract and epithelial-mesenchymal interactions are being clarified. In searching for extrinsic developmental regulators, numerous candidate trophic factors have been proposed, but compelling evidence remains elusive. A critical gene that initiates pancreas development has been identified, as well as a number of genes regulating liver, stomach, and intestinal development. Mutations in genes affecting neural crest cell migration have been shown to give rise to Hirschsprung's disease. Considerable progress has been achieved in understanding specific phenomena, such as the transcription factors regulating expression of sucrase-isomaltase and fatty acid-binding protein. The challenge for the future is to integrate these data into a more complete understanding of the physiology of gastrointestinal development.
Aging↗
Gene therapy for gastrointestinal disorders: just around the corner?
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Forecasting↗
Three distinct messenger RNA distribution patterns in human jejunal enterocytes.
BACKGROUND & AIMS: The importance of messenger RNA (mRNA) localization in human enterocytes is poorly understood. Previous studies from our laboratory have indicated that mRNAs are asymmetrically distributed in human intestinal epithelial cells, but in general colocalized with their encoded proteins. The aim of this study was to characterize, in human enterocytes, mRNA localization patterns of three genes with distinctly different functions. METHODS: mRNA distribution was determined by in situ hybridization with digoxigenin-labeled RNA probes in tissue sections of human jejunum. RESULTS: The mRNA for villin, a well-characterized microvillus cytoskeletal protein, was sorted to the basal region of the enterocyte. The mRNA for human sodium glucose cotransporter 1 was localized to the apical region, and the mRNA for human liver fatty acid-binding protein was distributed diffusely in the cytoplasm. CONCLUSIONS: The three distinct mRNA distribution patterns suggest that active mRNA sorting mechanisms exist in human enterocytes. This study also reveals for the first time that dichotomies may occur between the distribution patterns of sorted mRNAs and their encoded proteins.
Adult↗
Asymmetrical localization of mRNAs in enterocytes of human jejunum.
Intracellular localization of specific mRNAs is known to be a mechanism for targeting proteins to specific sites within the cell. Previous studies from this laboratory have demonstrated co-localization of mRNAs and proteins for a number of genes in absorptive enterocytes of fetal rat intestine. The present study was undertaken to examine in human enterocytes the intracellular localization patterns of mRNAs for the microvillous membrane proteins lactase-phlorizin hydrolase (LPH), sucrase-isomaltase (SI), and intestinal alkaline phosphatase (IAP), and the cytoskeletal protein beta-actin. In sections of human jejunum, mRNAs were localized by in situ hybridization using digoxigenin-labeled anti-sense RNA probes. Both LPH and SI mRNAs were localized to the apical region of villous enterocytes, whereas IAP and beta-actin mRNAs were detected both apically and basally relative to the nucleus. Therefore, in contrast to LPH, SI, and beta-actin mRNAs, which co-localize with their encoded proteins, that of IAP is present in the basal region of the cell where IAP protein has not directly been demonstrated to be present. Absorptive enterocytes from humans possess the mechanisms for intracellular mRNA localization, but not all mRNAs co-localize with their encoded proteins.
Actins↗
Total intestinal lactase and sucrase activities are reduced in aged rats.
Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are intestinal microvillus membrane hydrolases that play important roles in carbohydrate digestion. Although the expression of these enzymes during postnatal development has been characterized, the effect of old age on disaccharidase activity is poorly understood. In the present investigation, we examined the effect of aging on lactase and sucrase activities and their mRNA levels in the small intestines of 3-, 12- and 24- mo-old rats by sampling from nine equidistant segments of small intestine. Total intestinal disaccharidase activity or mRNA abundance was determined from areas under the proximal-to-distal curves. Rats 24 mo of age had total intestinal lactase and sucrase activities that were 12 and 38% lower, respectively, than the 3-mo-old animals (P < 0.05). In contrast, total LPH and SI mRNA abundance did not change significantly. Thus, total intestinal lactase and sucrase activities decrease with age in a manner that likely involves a posttranscriptional process. The age-related decline in disaccharidase activity, if extrapolated to humans, may have important implications for the digestion of carbohydrate contained in the diet of the elderly.
Aging↗
Increased C/EBP in fetal rat small intestine precedes initiation of differentiation marker mRNA synthesis.
Morphogenesis, initiation of differentiation marker gene expression, and their correlation with CCAT/enhancer binding protein (C/EBP) expression were analyzed in the developing fetal rat small intestine. Expressions of mRNAs for lactase-phlorizin hydrolase (LPH), intestinal alkaline phosphatase (IALP), carbamoyl-phosphate synthetase (CPS), and three isoforms of C/EBP were simultaneously determined by Northern blot analysis from 15 to 19 days of gestation. At 17 days of gestation, prior to villus formation as demonstrated by light and electron microscopy, only CPS and C/EBPalpha, -beta, and -delta expression could clearly be detected. Both LPH and IALP mRNA were definitely detectable in proximal and middle intestine on day 18, as soon as the stratified epithelium of the early intestine had been transformed into a single layer of columnar epithelium lining villi. This distribution was confirmed by in situ hybridization for LPH mRNA. During the period of transformation when the columnar epithelium and villi were forming, no LPH or IALP mRNA was detectable in the immature distal one-third of the fetal intestine. Preceding villus morphogenesis, immunostaining demonstrated nuclear localization of C/EBPalpha protein in intestinal epithelial cells, with continued expression in all enterocytes through 19 days of gestation. Enhanced expression of C/EBPalpha mRNA and protein began 24 h prior to the initiation of the differentiation markers, suggesting that it may play a role in regulation of fetal intestinal differentiation.
Alkaline Phosphatase↗
Quantitative analysis of lactase-phlorizin hydrolase expression in the absorptive enterocytes of newborn rat small intestine.
At birth, the mammalian small intestine displays regional differences in morphology as well as complex proximal-to-distal (horizontal) patterns of protein distribution. Lactase-phlorizin hydrolase (LPH), an enterocyte-specific disaccharidase crucial for the digestion of lactose in milk, reveals a characteristic horizontal pattern of expression at birth. However, it is not certain whether this topographic pattern is due to variations in epithelial structure along the length of the small intestine or to regional differences in the transcription of the LPH gene. In order to understand the mechanisms that regulate the regionalization of LPH at birth, we characterized the epithelial structure along the horizontal axis using stereologic techniques and correlated these data with the patterns of lactase activity and LPH mRNA abundance in the small intestine of unsuckled, newborn rats. Epithelial volume and microvillar surface area per unit of intestinal length decreased three-to fourfold from duodenum to distal ileum. In contrast, lactase activity and LPH mRNA abundance were highest in proximal jejunum and lowest in the most proximal and distal ends of the small intestine. Mean lactase activity per cell in proximal duodenum, proximal jejunum, and distal ileum was estimated at 12.0, 26.7, and 5.6 nU/absorptive enterocyte, respectively, and paralleled the concentration of LPH mRNA in the same segments: 20, 45, and 15 molecules of LPH mRNA/absorptive enterocyte. Our data indicate that horizontal gradients of lactase activity in the newborn rat intestine do not depend on epithelial organization or on enteral factors, since the horizontal gradient is established before suckling. Each absorptive enterocyte along the small intestine expresses lactase activity in a position-dependent manner which is controlled at the level of mRNA abundance.
Absorption↗
Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis.
BACKGROUND & AIMS: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS: These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.
Amino Acid Sequence↗
Restriction of lactase gene expression along the proximal-to-distal axis of rat small intestine occurs during postnatal development.
BACKGROUND/AIMS: Developmental changes of lactase activity along the proximal-to-distal axis of the small intestine are poorly understood. A study of delineate lactase gene expression at the cellular level was undertaken. METHODS: The topographical regulation of lactase was studied in conjunction with sucrase-isomaltase in proximal, middle, and distal segments of 0-, 7-, 14-, 16-, 18-, 21-, and 28-day-old and adult rats using in sity hybridization, immunohistochemistry, and ribonuclease protection assays. RESULTS: From 0 to 16 days, lactase messenger RNA (mRNA) and protein were abundant along the total length of the small intestine. However, at weaning, lactase mRNA and protein were no longer detectable in the terminal ileum. After 28 days, zones of reduced lactase expression were found in the duodenum and terminal ileum. These zones demonstrated expression of lactase protein in scattered enterocytes along the villus (patchy expression). In contrast, sucrase-isomaltase was first detected at 16 days, with patchy expression along the total small intestine; at 21 days it was abundant. CONCLUSIONS: Concordant changes in both lactase mRNA and protein detection during development suggest that the horizontal gradient of lactase enzyme expression is dependent on lactase mRNA abundance. Furthermore, zones of patchy lactase expression appear around weaning and flank the area of high lactase expression in the midintestine. Patchy expression is also found for sucrase-isomaltase before weaning.
Animals↗
Transcriptional regulation of intestinal hydrolase biosynthesis during postnatal development in rats.
Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are intestine-specific microvillus membrane hydrolases whose specific activities demonstrate reciprocal regulation during development but whose mechanisms of regulation have not been fully defined. To investigate transcriptional control of these two proteins, the rat LPH and SI genes were cloned, and antisense probes for preprocessed mRNAs (pre-mRNAs) were developed from intron sequence. LPH mRNA, as measured by quantitative ribonuclease (RNase) protection assays, was abundant before weaning and decreased two- to fourfold during weaning, whereas SI mRNA was first detected 14 days after birth and increased rapidly to abundant levels by age 28 days. LPH and SI pre-mRNA levels paralleled those of their respective mRNAs. LPH transcriptional rate declined during weaning, whereas that of SI increased during this time as determined by RNase protection assays of pre-mRNAs and nuclear run-on assays. In the adult rat, LPH mRNA was restricted to the jejunum and proximal ileum, whereas SI mRNA was detected throughout the small intestine, a pattern regulated by transcriptional rate as confirmed by nuclear run-on assays. Lactase and sucrase specific activities correlated well with their respective protein and mRNA concentrations in all experiments. We conclude that gene transcription plays a major role in the developmental and horizontal regulation of LPH and SI biosynthesis and that these two genes are regulated differently in rat small intestine.
Aging↗
Messenger RNA sorting in enterocytes. Co-localization with encoded proteins.
This study describes the intracellular compartmentalization of three different mRNAs in the polarized rat fetal enterocyte. They encode proteins that are known to be localized within different regions of the epithelial cell namely (i) the apical, membrane-bound glycoprotein, lactase-phlorizin hydrolase (lactase), (ii) the mitochondrially localized enzyme, carbamoylphosphate synthetase (CPS), and (iii) the cytoplasmically localized enzyme, phosphoenolpyruvate carboxykinase (PEPCK). These mRNAs are found in close proximity to their respective protein products, i.e. the apical membrane, mitochondria and cytoplasm, respectively. The significance of these observations is twofold; (i) they indicate that mRNAs are sorted into specific domains of the cytosol of intestinal epithelial cells; and (ii) they imply the presence of two distinct pathways of mRNA targeting one that allows transport of mRNAs that are translated on ribosomes associated with the rough endoplasmic reticulum (lactase mRNA), and the other that allows sorting of mRNAs that are translated on free polysomes (CPS and PEPCK mRNA).
Animals↗
Lactase gene expression during early development of rat small intestine.
Expression of lactase messenger (m) RNA and protein in rat small intestine during fetal and postnatal development was analyzed using in situ hybridization and immunohistochemistry. Lactase mRNA was first identified at 18 days of development, and lactase protein was first detected at day 20. Lactase mRNA and protein were present along the entire villus. Lactase mRNA increased, reaching a maximum at day 20. Just before birth a decrease in lactase mRNA was observed. In newborn intestine, lactase mRNA was present only from the base of the villus up to the mid-villus region and was undetectable up to the villus tips. Lactase protein continued to be expressed along the entire villus. These data show that expression of lactase mRNA and protein do not parallel, indicating a posttranscriptional control in fetal development. Lactase gene transcription is initiated late in gestation concomitant with villus formation and is exclusively seen in villus epithelial cells. The restriction after birth of lactase mRNA expression to cells at the villus base suggests the occurrence of a previously unknown step in postnatal differentiation of the enterocyte.
Age Factors↗
Molecular basis of lactase levels in adult humans.
The molecular basis of adult human "lactase deficiency" has long been a subject of controversy. To address this issue, small intestinal biopsies from orienta, black, and white patients were analyzed. Adjacent samples were assayed for lactase and sucrase specific activities and the sucrase/lactase ratio (high ratio signifies lactase deficiency), and the results were compared to lactase steady-state mRNA levels detected in Northern blots probed with a human lactase mDNA. All oriental patients had high ratios and no detectable lactase mRNA. Four black patients had a similar pattern; two with low ratios had detectable mRNA. The group of white patients displayed a range of findings, from high ratio/no mRNA to low ratio/considerable mRNA. Elevated levels of lactase mRNA always correlated with the presence of elevated levels of lactase enzyme activity, suggesting that the difference in levels of adult human intestinal lactase activity among racial groups may be regulated at the level of gene transcription.
Adult↗
Developmental regulation of the brush border hydrolase lactase.
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Animals↗
Cortisone and thyroxine modulate intestinal lactase and sucrase mRNA levels and activities in the suckling rat.
Glucocorticoids and thyroxine modulate postnatal intestinal sucrase and lactase activities. Whether changes in enzyme activity are accompanied by changes in enzyme mRNA levels were determined in day 6 rats given thyroxine, cortisone, or thyroxine plus cortisone and killed 3 days later. Cortisone induced precocious expression of jejunal sucrase activity which was enhanced when cortisone plus thyroxine was administered; sucrase mRNA changed in parallel. Jejunal lactase activity was unaffected by thyroxine and was increased after cortisone, but not after thyroxine plus cortisone. Jejunal lactase mRNA levels increased equally after cortisone or after cortisone plus thyroxine. Thus, cortisone induces coordinated increases in sucrase and lactase activities and in corresponding mRNA levels. Thyroxine only enhances cortisone induced sucrase expression and antagonizes cortisone by depressing lactase activity post-translationally.
Animals↗
Lactose intolerance and the genetic regulation of intestinal lactase-phlorizin hydrolase.
Lactase-phlorizin hydrolase, which hydrolyzes lactose, the major carbohydrate in milk, plays a critical role in the nutrition of the mammalian neonate. Lactose intolerance in adult humans is common, usually due to low levels of small intestinal lactase. Low lactase levels result from either intestinal injury or (in the majority of the world's adult population) alterations in the genetic expression of lactase. Although the mechanism of decreased lactase levels has been the subject of intensive investigation, no consensus has yet emerged. Recent studies have begun to define the cellular and molecular biology of this enzyme. In animals and humans, a glycosylated precursor is proteolytically cleaved to yield the mature enzyme on the microvillus membrane of the enterocyte, bound to the lipid bilayer only by a hydrophobic anchor sequence. The enzyme hydrolyzes lactose, phlorizin, and glycosylceramides. A decline in lactase specific activity occurs at the time of weaning in most mammalian species; in most humans who have low lactase activity as adults, the decline occurs at approximately 3-5 years of age. In a few human groups, the elevated juvenile level of lactase specific activity persists throughout adulthood. These developmental patterns of lactase expression are most likely regulated at the level of gene transcription.
Animals↗
Clinical aspects of lactose intolerance in children and adults.
The principal carbohydrate of human milk is the disaccharide lactose. In human and all mammalian species, lactose is hydrolyzed in the small intestine by lactase-phlorizin hydrolase, also abbreviated as lactase. The absence of lactase results in the passage of undigested lactose into the large intestine and is associated with a well-known clinical syndrome: lactose intolerance. Low lactase levels result either from intestinal injury or, as in the majority of world's adult population, from alterations in the genetic expression of lactase. In this review terminology, pathophysiology, symptoms, diagnostic procedures, and therapy of lactose intolerance will be discussed.
Adult↗