Sucrase-isomaltase mRNA Levels Research Articles

Molecular Regulations of Mucosal Maltase Expressions.

Two major α-glucosidase (maltase) genes, sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM), respectively, are expressed in the small intestine. In this review, we have summarized whether jejunal expression of these maltase genes is regulated by dietary manipulations, which may affect carbohydrate availability from the luminal side, through changes in the binding of transcription factors and/or histone code on these genes. Studies using a model of mice fed either a low-starch or a high-starch diet for 7 days, found the mRNA levels of SI, MGAM, and Na-glucose cotransporter (SGLT1) genes in the jejunum to be increased in parallel by feeding a high-starch diet. Chromatin immunoprecipitation assays, using jejunal tissue of mice and rats fed a high-starch diet, revealed that the diet increased the acetylations of histones H3 and H4, bindings of coactivators, including general control of amino acid synthesis (GCN5) and the transcriptional factors, including caudal-related homeobox 2 (CDX2), and hepatocyte nuclear factor 1 (HNF1), not only in the promoter/enhancer regions, but also in the transcribed regions of SI and MGAM genes. Feeding rats a diet rich in resistant starch led to a concomitant reduction of mRNA levels of the MGAM gene and histone H3 modifications (acetylations and di-/tri-methylations) in the jejunum. These data suggest that a signal elicited by available glucose in the jejunal mucosa is associated with SI and MGAM gene expressions through a histone code, such as acetylation and di-/tri-methylations of histone H3 in the promoter/enhancer and transcribed regions of SI and MGAM genes.

Dietary Sucrose Enhances Intestinal Lactase Gene Expression in Euthyroid Rats

It is postulated that dietary carbohydrates and thyroid hormones are major regulators for expression of the lactase/phlorizin hydrolase (LPH) gene in rat jejunum. In this study, we investigated the effects of thyroid hormones and dietary sucrose on LPH gene expression and lactase activity in starved rats. Firstly, animals at 8 wk of age were fed a low-starch diet (5.5% energy as cornstarch) or high-starch diet (71% energy as cornstarch) for 7 d (experiment 1). The mRNA level of LPH as well as lactase activity significantly decreased in rats fed the low-starch diet as compared to those fed the high-starch diet. To investigate the effects of thyroid hormone status, the animals previously fed the low-starch diet were starved for 3 d, and half of the animals were given intraperitoneal (i.p.) injections of 20 microg/ 100 g body weight triiodothyronine (T3) twice daily (experiment 2). The LPH mRNA level and lactase activity were elevated by starvation for 3 d, but they were repressed by the injection of T3 during starvation. To investigate the effects of dietary sucrose in starved rats, they were force-fed a sucrose diet for 6 h (experiment 3). The LPH gene expression and lactase activity were up-regulated by force-feeding a sucrose diet, only when the animals were kept in euthyroid status by daily T3 administrations. In contrast, the sucrase-isomaltase mRNA levels and sucrase activity were unaffected by force-feeding the sucrose diet for both T3-treated and untreated starved rats. Our work suggests that dietary sucrose is capable of enhancing lactase gene expression in starved rats when they have a sustainable thyroid hormone level.

Impairments in enzyme activity and biosynthesis of brush border-associated hydrolases in human intestinal Caco-2/TC7 cells infected by members of the Afa/Dr family of diffusely adhering Escherichia coli.

Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the cells. We report here that infection by Afa/Dr DAEC wild-type strains C1845 and IH11128 in polarized human fully differentiated Caco-2/TC7 cells dramatically impaired the enzyme activity of functional brush border-associated proteins sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPP IV). Blockers of the transduction signal molecules, previously found to be active against the Afa/Dr DAEC-induced cytoskeleton injury, were inactive against the Afa/Dr-induced decrease in sucrase enzyme activity. In parallel, Afa/Dr DAEC infection promotes the blockade of the biosynthesis of SI and DPP IV without affection enzyme stability. The observation that no changes occurred in mRNA levels of SI and DPP IV upon infection suggested that the decrease in biosynthesis probably resulted from a decrease in the translation rate. When the cells were infected with recombinant E. coli strains expressing homologous adhesins of the wild-type strains, neither a decrease in sucrase and DPP IV enzyme activities nor an inhibition of enzyme biosynthesis were observed. In conclusion, taken together, these data give new insights into the mechanisms by which the wild-type Afa/Dr DAEC strains induce functional injuries in polarized fully differentiated human intestinal cells. Moreover, the results revealed that other pathogenic factor(s) distinct from the Afa/Dr adhesins may play(s) a crucial role in this mechanism of pathogenicity.

Regulation of the expression of carbohydrate digestion/absorption-related genes.

To explore the underlying molecular mechanism whereby nutrients modulate the expression of intestinal digestion/absorption-related genes, we have cloned the 5' flanking regions of two representing disaccharidase genes, i.e. sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH), and investigated whether the binding activity of putative common nuclear factor(s) binding to the cis-elements located in these regions is altered by dietary manipulations. Oro-gastric feeding of a sucrose-containing diet to rats caused parallel increases in SI mRNA and LPH mRNA levels within 3 h. Among the monosaccharides tested, fructose gave rise to the most prominent increase in the mRNA levels of SI and LPH genes, which were accompanied by a coordinate rise in the mRNA levels of two microvillar hexose transporters, i.e. SGLT1 and GLUT5. Nuclear run-on assays revealed that fructose, but not glucose, increased the transcription of SI, LPH and GLUT5. DNase I footprinting analysis of the rat LPH gene showed that the protected region conserved the same sequence as the cis-element (CE-LPH1) reported in the pig LPH gene. Electrophoretic mobility shift assay using CE-LPH1 and the related cis-element of SI gene (SIF1) revealed that nuclear extracts from the jejunum of rats fed the high-starch diet gave greater density of retarded bands than those of rats fed the low-starch diet. Force feeding a fructose diet gave rise to an increase in the binding of the dimeric nuclear protein (Cdx-2) to the SIF1 element. These results suggest that the cis-elements of CE-LPH1 and SIF1 might be involved in the carbohydrate-induced increases of the transcription of LPH and SI, presumably through a change in the expression and/or binding activity of Cdx-2.

Lactase-Phlorizin Hydrolase and Sucrase-Isomaltase Genes Are Expressed Differently Along the Villus-Crypt Axis of Rat Jejunum

Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are two disaccharidases specifically expressed in small intestinal absorptive cells. We previously showed that the transcripts of both genes are elevated within 12 h of carbohydrate intake. To examine at which locus of villus-crypt axis this response to dietary carbohydrate occurs, 6-wk-old rats were fed a low-carbohydrate diet (5% energy) for 7 d, and then force-fed either the low-carbohydrate diet or a sucrose (40% energy) diet during the last 6 h. Cryostat sectioning of jejunal segments followed by RNA blot hybridizations of the transcripts revealed that, unlike SI mRNA which was expressed maximally in the lower villus, maximal LPH mRNA level was attained at the upper villus. The distribution of the respective immunoreactive protein and the enzymatic activity was shifted more toward the villus tips for LPH than for SI. Force-feeding the sucrose diet caused an abrupt increase in SI mRNA level in the lower villus within 3 h, while the rise in LPH mRNA level occurred in the mid- and upper-villus. The diet-induced increases in the LPH mRNA and SI mRNA levels were abolished along the entire villus by the administration of actinomycin D. These results suggest that LPH gene is maximally expressed in more apical villus cells than SI gene, and that dietary sucrose elicits enhancement of the gene expressions in the villus cells which are accumulating the respective transcripts.

Dissociation between growth arrest and differentiation in Caco-2 subclone expressing high levels of sucrase.

Growth arrest and cell differentiation are generally considered temporally and functionally linked phenomena in small intestinal crypt cells and colon tumor cell lines (Caco-2, HT-29). We have derived a Caco-2 subclone (NGI3) that deviates from such a paradigm. In striking contrast with the parental cells, proliferative and subconfluent NGI3 cells were found to express sucrase-isomaltase (SI) mRNA and to synthesize relatively high levels of SI, dipeptidyl peptidase IV, and aminopeptidase N (APN). In postconfluent cells, little difference was seen in SI mRNA levels between Caco-2 and NGI3 cells, but the latter still expressed much higher levels of SI that could be attributed to higher rates of translation. APN expression was also greatly enhanced in NGI3 cells. To determine whether high levels of brush-border enzymes correlated with expression of cell-cycle regulatory proteins, we investigated their relative cellular levels in growing and growth-arrested cells. The results showed that the cyclin-dependent kinase inhibitors (p21 and p27) and D-type cyclins (D1 and D3) were all induced in postconfluent cells, but NGI3 cells expressed much higher levels of p21. This study demonstrated that cell growth and expression of differentiated traits are not mutually exclusive in intestinal epithelial cells and provided evidence indicating that posttranscriptional events play an important role in regulation of SI expression.

Sucrase-Isomaltase and Hexose Transporter Gene Expressions Are Coordinately Enhanced by Dietary Fructose in Rat Jejunum

We previously demonstrated that the levels of mRNAs of both sucrase-isomaltase (SI) and sodium/D-glucose transporter (SGLT1) are modulated by dietary sucrose in the rat jejunum. In the present study, we investigated whether the transcription of the gene coding SI is regulated by certain types of monosaccharides. Force-feeding a fructose and sucrose diet, (40% energy as fructose or sucrose) gave rise to parallel increases in the transcripts of SI and intestinal hexose transporters (SGLT1, GLUT5, and GLUT2) within 12 h. Force-feeding a glycerol-containing diet also caused an enhancement of SI, SGLT1, and GLUT2 mRNA levels. However, feeding the diet containing glucose or α-methylglucoside generally did not increase the transcript levels of SI or the intestinal hexose transporters. Nuclear run-on assays revealed that fructose as well as sucrose increased the transcription of both SI and GLUT5 genes and that the transcription rates of these genes were unaffected by glucose. These results suggest that fructose (or a metabolite) is capable of increasing the mRNA levels of SI and hexose transporters in the small intestine and that transcriptional regulation might play a pivotal role in the carbohydrate-induced coordinate enhancement of SI and fructose transporter gene expression

Glucose-dependent transcriptional regulation of the human sucrase-isomaltase (SI) gene

We have previously shown that the transcription of the human sucrase-isomaltase (SI) gene was negatively regulated by glucose. Using two clonal metabolic variants of the human colon adenocarcinoma cell line Caco-2 we demonstrate here that: 1) although similar growth-related variations of phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-diphosphatase (F1,6-dPase), pyruvate kinase (PK) and SI mRNA levels are observed, only F1,6-dPase, PK and SI mRNA levels vary in the same way in response to modifications of glucose utilization; and 2) regulatory elements responsible for the glucose-dependent transcription of the SI gene are located within the −370/+30 region of the promoter.

Cell cycle protein suppression and p21 induction in differentiating Caco-2 cells.

Despite intensive efforts, the exact cellular mechanisms leading to gut differentiation and development remain largely undefined. The cyclins, the cyclin-dependent kinases (Cdks), and the Cdk inhibitors (e.g., p21 and p27) are proteins that are important for cell cycle progression, subsequent growth inhibition, and differentiation of various cell types. The purpose of our study was to better define the role of these cell cycle proteins in gut differentiation using the Caco-2 human cell line, which spontaneously differentiates to a small bowel phenotype, as demonstrated by induction of sucrase-isomaltase (SI) gene expression. We found that protein levels of the cyclins (both D- and E-type) and the Cdks (both Cdk2 and Cdk4) progressively decreased in postconfluent Caco-2 cells. Moreover, cyclin E-associated histone H1 kinase activity decreased in an analogous fashion as the cyclins and Cdks. In contrast, induction of the Cdk inhibitor p21 occurred by 3 days postconfluency, which was before the increase in SI mRNA levels. These changes in the cell cycle proteins, which include a progressive decrease of the cyclins and Cdks and a concomitant induction of p21, suggest an important role for these proteins in Caco-2 cell differentiation. Identifying the cell cycle mechanisms responsible for intestinal cell differentiation will be important to our understanding of both normal gut development as well as gut neoplasia, which involves aberrant regulation of cell cycle arrest.

Dietary regulation of sucrase-isomaltase gene expression in rat jejunum

We have previously demonstrated that intake of fat as well as carbohydrate affects the activity and immunoreactive amount of sucrase-isomaltase (S-I) in rat jejunum. To examine whether diet-related changes in sucrase and isomaltase activities are accompanied by the variations of sucrase-isomaltase mRNA levels, 7-week-old rats were fed either a high-long-chain triacylglycerols diet (73 energy% as corn oil), a high-medium-chain triacylglycerols (MCT) diet (66 energy% as MCT, 7 energy% as corn oil) or a high-carbohydrate diet (70 energy% as corn starch) for 7 days. Northern blot analysis revealed that S-I mRNA levels were abundant in the jejunum of rats fed the high-MCT diet; the levels were similar to those in the rats fed the high-carbohydrate diet. Force-feeding a high-sucrose diet (40 energy% as sucrose) brought about a parallel rise in both S-I mRNA and sodium/ d-glucose cotransporter (SGLT1) mRNA levels within 12 h. Force-feeding the high-MCT diet also produced an elevation of S-I mRNA and SGLT1 mRNA. However, force-feeding a diet containing α-methylglucoside, a non-metabolizable but actively transported sugar, did not increase S-I mRNA or SGLT1 mRNA level; sucrase activity was nevertheless elevated by feeding α-methylglucoside diet. These results suggest that not only carbohydrate intake but also MCT intake might influence S-I mRNA and SGLT1 mRNA levels in the jejunum, presumably through common metabolite(s) of carbohydrates and MCT, and that carbohydrate may play another role in enhancement of the sucrase activity through modulation of translation and/or posttranslational modifications of the sucrase-isomaltase complex.

Inhibition of sucrose-isomaltase expression by EGF in the human colon adenocarcinoma cells Caco-2

To investigate the role and mechanism of action of epidermal growth factor (EGF) in the intestinal epithelium, we have studied its influence on proliferation and differentiation of Caco-2 cells, a human colon adenocarcinoma cell line exhibiting several characteristics of adult small intestinal enterocytes. A clone of Caco-2 cells synthesizing minimal amounts of transforming growth factor-alpha (TGF-alpha)/epidermal growth factor (EGF)-like activity was used in these studies. Cells grown in the presence of 20-200 ng EGF/ml exhibited increased DNA synthesis and proliferation; formation of morphologically poorly differentiated multilayers was observed at 200 ng EGF/ml. At all concentrations tested EGF produced a significant and marked reduction in sucrase activity, whereas other brush-border enzymes (aminopeptidase N, alkaline phosphatase, dipeptidylpeptidase IV) were only marginally affected. EGF influenced sucrase expression at two different levels. At 20 ng/ml, it affected primarily sucrase-isomaltase processing in the endoplasmic reticulum and/or increased its degradation. At 200 ng EGF/ml, a significant and marked reduction in sucrase-isomaltase mRNA levels and biosynthesis was observed. These results demonstrated that EGF has important and selective effects on Caco-2 cell proliferation and differentiation and may affect different cellular activities depending on its concentration.

Determinants of regional sucrase-isomaltase expression in adult rat small intestine.

Sucrase-isomaltase (SI) expression along the longitudinal and vertical axis of the small intestine was studied by sequentially isolating enterocytes from villus to crypt of rat proximal jejunum and distal ileum. Gradients of sucrase activity were observed with greatest activity occurring in jejunal and villus regions. Along the villus-to-crypt axis, gradients of SI mRNA abundance corresponded with activity. However, along the longitudinal axis no differences in SI mRNA levels were observed, thus not accounting for the observed 3-5-fold difference in SI activities between jejunum and ileum. Comparison of SI immunoprecipitates from jejunal and ileal mucosal scrapings showed significant differences in gel mobilities of the more mature forms, which did not appear to affect SI functional activities. When relative rates of de novo SI protein synthesis were compared, [35S]methionine incorporation into all SI forms was observed to be 3-5-fold greater in jejunum than in ileum at all time points. Because these results suggested differences in regional translational regulation, subcellular distribution of SI mRNA in jejunal and ileal epithelial cells was compared. A greater proportion of jejunal SI mRNA was found to be associated with membrane-bound polyribosomes. We conclude 1) sucrase expression along the villus-to-crypt axis correlates with SI mRNA abundance, 2) post-translational processing of SI differ in ileum and jejunum, but appear not to determine SI expression, and 3) differences in translational processing in distal ileum and proximal jejunum may determine sucrase activity along the longitudinal axis of rat small intestine.

Molecular cloning and characterization of a rat intestinal sucrase-isomaltase cDNA. Regulation of sucrase-isomaltase gene expression by sucrose feeding

To investigate the regulation of expression of intestinal sucrase-isomaltase (SI) complex in response to sucrose feeding, we isolated a cDNA (RPSI 1) encoding partially the pro-SI of rat intestinal mucosa. The clone consists of 1929 mRNA-derived nucleotides, which covered the region including the C-terminal part of the isomaltase and the N-terminal part of the sucrase in the final SI complex. Nucleotide and amino-acid sequences of RPSI 1 were compared with their corresponding regions in rabbit pro-SI. A greater similarity was found in sucrase parts than in isomaltase parts of the two species. Northern blot analysis revealed that the mRNA levels of SI increased rapidly after sucrose force feeding, while those of rats fed a carbohydrate-free diet did not. These changes in mRNA levels correlated with the corresponding enzyme activities. The results demonstrate that the induction of SI activities is directly associated with an increase in SI mRNA levels. Our results also suggest that circadian modulation of SI transcription may occur in basic SI gene expression.

Reversible forskolin-induced impairment of sucrase-isomaltase mRNA levels, biosynthesis, and transport to the brush border membrane in Caco-2 cells.

Hybridization analysis of mRNA with a cDNA probe for human sucrase-isomaltase, pulse-chase experiments with L-[35S]-methionine followed by SDS-PAGE, and immunofluorescence detection of sucrase-isomaltase were used to analyze the level(s) at which forskolin interferes with the expression of the enzyme in Caco-2 cells in culture. Three effects are observed in Caco-2 cells treated with forskolin: 1) a marked decrease in the level of sucrase-isomaltase mRNA, 2) a marked decrease in the biosynthesis of the enzyme without any alteration of its stability, and 3) an almost total inhibition of its transport to the brush border membrane. All three effects are reversible when the drug is removed from the culture medium, though this reversibility is asynchronous: transport to the brush border membrane resumes after 24 h, sucrase-isomaltase mRNA levels are back to the normal after 5 days, whereas the biosynthesis of the enzyme, although increasing progressively, remains lower than in control cells, even 10 days after removal of the drug. The possibility that some effects are directly dependent on cAMP and others a consequence of changes in glucose metabolism is discussed.