Oral polyamine administration modifies the ontogeny of hexose transporter gene expression in the postnatal rat intestine.

Gastrointestinal mucosal polyamines influence enterocyte proliferation and differentiation during small intestinal maturation in the rat. Studies in postnatal rats have shown that ornithine decarboxylase (ODC) protein and mRNA peak before the maximal expression of brush-border membrane (BBM) sucrase-isomaltase (SI) and the sugar transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2). This study was undertaken to test the hypothesis that the oral administration of spermidine in postnatal rats upregulates the expression of ODC, thereby enhancing the expression of SI and SGLT1 in the brush-border membrane as well as basolateral membrane-facilitative GLUT2 and Na(+)-K(+)-ATPase. Northern and Western blot analyses were performed with antibodies and cDNA probes specific for SI, SGLT1, GLUT2, alpha(1)- and beta(1)-subunits of Na(+)-K(+)-ATPase, and ODC. Postnatal rats fed 6 mumol spermidine daily for 3 days from days 7 to 9 were killed either on postnatal day 10 (Sp10) or day 13 following a 3-day washout period (Sp13). Sp10 rats showed a precocious increase in the abundance of mRNAs for SI, SGLT1, and GLUT2 and Na(+)-K(+)-ATPase activity and alpha(1)- and beta(1)-isoform gene expression compared with controls. ODC activity and protein and mRNA abundance were also increased in Sp10 animals. The increased expression of these genes was not sustained in Sp13 rats, suggesting that these effects were transient. Thus, 3 days of oral polyamine administration induces the precocious maturation of glucose transporters in the postnatal rat small intestine, which may be mediated by alterations in ODC expression.

Gata4 and Hnf1alpha are partially required for the expression of specific intestinal genes during development.

The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions. With the use of gene inactivation models in mature mouse jejunum, we have previously shown that a member of the zinc finger transcription factor family (Gata4) and hepatocyte nuclear factor-1alpha (Hnf1alpha) are each indispensable for LPH and Fabp1 gene expression but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1alpha regulate LPH, Fabp1, and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1alpha resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1alpha had a partial effect ( approximately 50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1alpha protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of their mRNAs. Finally, we show that neither Gata4 nor Hnf1alpha mediates the glucocorticoid-induced precocious maturation of the intestine but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1alpha that are dependent on the developmental time frame in which they are expressed.

HNF-1alpha participates in glucose regulation of sucrase-isomaltase gene expression in epithelial intestinal cells.

Sucrase-isomaltase (SI) gene expression is negatively regulated by glucose, but its molecular mechanism is not completely clear. The purpose of this study is to investigate whether HNF-1alpha and HNF-1beta contribute to glucose regulation of SI gene expression. To explore this question, we examined the association of gene expressions between SI and HNF-1alpha and HNF-1beta in Caco-2 cells cultured in medium containing 2.0 and 16.7 mM glucose. We found that gene expression of HNF-1alpha but not HNF-1beta exhibits a positive correlation with that of SI regulated by glucose. Moreover, to elucidate whether glucose regulation of SI gene expression is changed when HNF-1alpha and HNF-1beta are inhibited, we produced three stable cell lines, in which dominant-negative mutant HNF-1alphaT539fsdelC, mutant HNF-1betaR177X, and empty vector (as a control), respectively, were stably expressed. We found that the glucose regulation of SI gene expression was significantly attenuated in HNF-1alphaT539fsdelC cells, but it was well maintained in empty vector and HNF-1betaR177X cells. These results suggest that HNF-1alpha participates in glucose regulation of SI gene expression.

Concomitant cell growth and differentiation are dependent on erbB1 and integrin activation in an autonomously surviving colon adenocarcinoma: involvement of autocrine amphiregulin secretion.

In normal colon epithelium, cell proliferation is followed by cell differentiation. The purpose of this work was to investigate, in the HT29-D4 colon adenocarcinoma cell line, the occurrence of a temporal sequence of changes in cell proliferation and differentiation, the role of autocrine EGF family ligands and to determine which transduction pathway(s) are involved in these processes. In a medium lacking both growth factor and serum, HT29-D4 cells secreted amphiregulin (AR), which was shown to be strongly involved in cell adhesion, growth and differentiation. In the main, integrins alpha2beta1 and alphavbeta6 intervened in these processes. Using tyrphostins, it was demonstrated that AR involvement was mediated through the ErbB1/ERK1,2 and ErbB1/FAK pathways. These signalling molecules were directly involved in pRb inhibition and, thus, in cyclin A expression. Concomitantly, colon differentiation markers were also expressed. Furthermore, terminal cell maturation resulted in a colon absorptive cell with strong polarisation, the growth of which was inhibited by tyrphostin and an ERK1,2 inhibitor. It was concluded that in a colon adenocarcinoma, cell proliferation and differentiation can occur concomitantly and that these deregulated processes are controlled by autocrine secretion through the ErbB1/ERK1,2 and FAK pathways.

A cyclic AMP protein kinase A-dependent mechanism by which rotavirus impairs the expression and enzyme activity of brush border-associated sucrase-isomaltase in differentiated intestinal Caco-2 cells.

We undertook a study of the mechanism by which rhesus monkey rotavirus (RRV) impairs the expression and enzyme activity of brush border-associated sucrase isomaltase (SI) in cultured, human, fully differentiated, intestinal Caco-2 cells. We provide evidence that the RRV-induced defects in the expression and enzyme activity of SI are not related to the previously observed, RRV-induced, Ca2+ -dependent, disassembly of the F-actin cytoskeleton. This conclusion is based on the facts that: (i) the intracellular Ca2+ blocker, BAPTA/AM, which antagonizes the RRV-induced increase in [Ca2+](i), fails to inhibit the RRV-induced decrease in SI expression and enzyme activity; and (ii) Jasplakinolide (JAS) treatment, known to stabilize actin filaments, had no effect on the RRV-induced decrease in SI expression. Results reported here demonstrate that the RRV-induced impairment in the expression and enzyme activity of brush border-associated SI results from a hitherto unknown mechanism involving PKA signalling. This conclusion is based on the observations that (i) intracellular cAMP was increased in RRV-infected cells and (ii) treatment of RRV-infected cells with PKA blockers resulted in the reappearance of apical SI expression, accompanied by the restoration of the enzyme activity at the brush border. In addition, in RRV-infected cells a twofold increase of phosphorylated form of cytokeratin 18 was observed after immunopurification and Western Blot analysis, which was antagonized by exposing the RRV-infected cells to the PKA blockers.

Morphological changes and increased sucrase and isomaltase activity in small intestines of insulin-deficient and type 2 diabetic rats.

The small intestine plays an important role in the digestion and absorption of many nutrients. To investigate the contribution of carbohydrate digestion to diabetes mellitus, we examined the morphological changes of the small intestine, and the expression of sucrase-isomaltase, which is one of the intestinal disaccharidases, in diabetic model rat, that is the streptozotocin-induced (STZ) diabetic rat (insulin-deficient model), and the Otsuka Long-Evans Tokushima Fatty (OLETF) rats and the Goto-Kakizaki (GK) rats (type 2 diabetic models). Intestinal hyperplasia was observed in STZ, OLETF, and GK rats. Moreover, in the small intestine of each diabetic strain, the proliferating cell nuclear antigen (PCNA)-labeling index, which is a marker of proliferation, was higher than in the respective control. Cdx1 and Cdx2, known to be transcriptional factors related to intestinal proliferation and differentiation, were more highly expressed in STZ, OLETF and GK rats than in the respective controls. These findings indicate that small intestinal hyperplasia, and thereby the resultant increase of total activity of disaccharidases such as sucrase and isomaltase in the entire small intestine, might be one of the reasons for postprandial hyperglycemia in diabetes mellitus.

Enterocytic columnar non-goblet cells of Barrett's esophagus--an immunohistochemical demonstration of association with malignant evolution.

Barrett's epithelium (BE), the esophageal columnar-lining with intestinal differentiation, is a premalignant condition predisposing to adenocarcinoma. Columnar cells are the prevalent element of BE, but the hallmark of intestinal differentiation is the goblet population that defines the specialised columnar epithelium (SCE). We have demonstrated that columnar cells adjacent to Barrett's adenocarcinoma (BA) exhibit enterocytic features in areas with and without SCE. Nevertheless, the relationship between malignancy and the presence of these elements is not established. To investigate whether intestinal differentiated cells, other than goblet cells, are associated to neoplasia we compared the prevalence of enterocytic features in columnar elements with and without associated BA through the use of sucrase-isomaltase (SI) immunoreactivity in 31 columnar esophageal segments (CLES) and 12 BA. In metaplasia, SI was only expressed at the columnar cells. Apical staining was exclusive of CLES with SCE. SI was present at the cytoplasm in 22.2% of CLES without SCE. Apical SI occurred in BE with and without carcinoma, similarly in areas with and without SCE (p = 0.11 and p = 0.50, respectively). In areas with SCE, columnar cells with apical SI were more frequent in cases of BE adjacent to carcinoma than in cases without neoplasia but the difference did not reach significance (p = 0.053). In areas without SCE, apical SI was significantly (p = 0.01) more frequent in cases with carcinoma. Apical SI was equally found in neoplastic as in metaplastic areas, with and without SCE, (p = 0.07 and p = 0.40, respectively). In conclusion this study on the frequency of SI on CLES with and without neoplasia demonstrated that additionally to SCE, metaplastic enterocytic cells are also associated with malignancy. It also confirmed that the presence of intestinal features are underestimated if only goblet elements are used for its identification, reinforcing the utility of the immunohistochemical recognition of enterocytic characteristics for establishing the diagnosis of BE.

A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP.

Intestinal gene regulation involves mechanisms that direct temporal expression along the vertical and horizontal axes of the alimentary tract. Sucrase-isomaltase (SI), the product of an enterocyte-specific gene, exhibits a complex pattern of expression. Generation of transgenic mice with a mutated SI transgene showed involvement of an overlapping CDP (CCAAT displacement protein)-GATA element in colonic repression of SI throughout postnatal intestinal development. We define this element as CRESIP (colon-repressive element of the SI promoter). Cux/CDP interacts with SI and represses SI promoter activity in a CRESIP-dependent manner. Cux/CDP homozygous mutant mice displayed increased expression of SI mRNA during early postnatal development. Our results demonstrate that an intestinal gene can be repressed in the distal gut and identify Cux/CDP as a regulator of this repression during development.

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.

Sucrase-alpha-dextrinase in the spontaneously diabetic BioBreed Wistar rat: altered intracellular carbohydrate processing.

Sucrase-alpha-dextrinase (S-D), a glycoprotein hydrolase in the border surface of the enterocyte, is altered in congenitally diabetic BioBreed Wistar (BB(d)) rats. Its intracellular assembly was examined in the current studies. Following pulse-chase experiments, S--D was specifically immuno-isolated from ER-Golgi and brush border membranes, and examined by SDS-PAGE and autoradiography. While synthesis and co-translational glycosylation of an immature species, P(i), in the ER proceeded normally, post-translational maturation of the N-linked carbohydrate chains was altered in the BB(d) rat. The mature species, P(m), was 10 kDa larger in BB(d) than in normal rats, and approximately 25% of its N-linked chains remained immature. The difference in mass was attributed to an appreciably larger mass of the O-linked chains of P(m) in BB(d) rats. In vivo kinetics of intracellular assembly displayed a delay in the appearance of P(m) in Golgi (Wistar, 15 min; BB(d), 30--60 min). These experiments, revealing structural alterations in congenital diabetes suggest an important role for intracellular glycosylation in the orderly assembly and processing of brush border S-D in the enterocyte.

Non-goblet cell population of Barrett's esophagus: an immunohistochemical demonstration of intestinal differentiation.

Barrett's esophagus develops with the following 2 distinct types of lining mucosa: with and without specialized intestinal metaplasia (SIM). Goblet cells found only in SIM areas identify an intestinal phenotype, recognized as the histological hallmark diagnosing Barrett's metaplasia, and selecting high-risk patients for endoscopic surveillance. The columnar non-goblet cells are the major component of the heterogeneous Barrett's metaplastic cell population and are present in areas either with or without SIM. Their significance in the differentiation of columnar-lined esophagus, and their relationship to malignancy, is still unclear. This immunohistochemical study used two markers of enterocytic differentiation, to explore the intestinal phenotype of the non-goblet cell population of Barrett's epithelium and Barrett's-associated adenocarcinoma cells. Sucrase-isomaltase (SI) and dipeptidilpeptidase IV (DPP) immunoexpression was assessed in paraffin-embedded samples of 12 surgical specimens containing Barrett's esophageal mucosa in association with adenocarcinoma/high grade dysplasia. Ileal mucosa and mucosa from normal gastric and esophageal segments of the surgical specimen were used as positive and negative controls, respectively. SI and DPP were expressed by the neoplastic cells and the columnar non-goblet, being negative in goblet cells. The localization of the enzymes was predominantly apical for SI and cytoplasmatic for DPP. There was immunoreactivity for SI in 58.3% of the carcinomas and in 66.6% of Barrett's mucosa, with equal frequency in areas with and without SIM. DPP was identified in 66.6% of the carcinomas, in 50% of the cases of Barrett's metaplasia with SIM, and in 75% of those without SIM. The columnar non-goblet cell components of Barrett's metaplasia contain small intestine enzymes in the areas either with or without SIM, which suggests that they identify an "incomplete form" of intestinal metaplasia. The demonstration that the two enzymes, SI and DPP, are produced by the columnar non-goblet cell metaplastic population and by the neoplastic cells of the associated adenocarcinoma, indicates that, in addition to the goblet cells, the non-goblet elements may also be involved in the malignant transformation of Barrett's esophagus.

Disordered expression of the sucrase-isomaltase complex in the small intestine in Otsuka Long-Evans tokushima fatty rats, a model of non-insulin-dependent diabetes mellitus with insulin resistance.

To clarify the relationship between diabetes mellitus and carbohydrate digestion, the activities of sucrase and isomaltase, which form a complex enzyme (SI complex) on the brush border membranes, were compared in the progression of diabetes mellitus in Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of human non-insulin-dependent diabetes mellitus with insulin resistance, and Long-Evans Tokushima Otsuka (LETO) rats as non-diabetic controls. Until 40 weeks of age, OLETF rats were obese and had a high plasma glucose level, compared to age-matched LETO rats, but the sucrase and isomaltase activities showed no significant differences between the two strains. Oral glucose tolerance test revealed that during 40-48 weeks of age, NIDDM became very severe with advancing insulin resistance in OLETF rats. In OLETF rats, in contrast to LETO rats, at 48 weeks of age, abnormal increases in the sucrase and isomaltase activities occurred, along with a remarkable decrease in body weight and a further great increase in the plasma glucose level in the non-fasting state. Hyperinsulinemia occurred in 20-week-old OLETF rats; however, at 40 and 48 weeks of age, the plasma insulin level in the non-fasting state in OLETF rats was not significantly different from that in LETO rats. The level of mRNA encoding the SI complex increased abnormally in 48-week-old OLETF rats. These results suggest that the advance of insulin resistance leads to an increase in the expression of the SI complex on the transcriptional level.

Keratinocyte growth factor down-regulates expression of the sucrase-isomaltase gene in Caco-2 intestinal epithelial cells.

The molecular mechanisms that regulate the proliferation and differentiation of intestinal mucosal epithelial cells are not well understood. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth factor that may be involved in the maintenance of mucosal epithelial populations and in mediating epithelial repair after injury. The sucrase-isomaltase (SI) gene, which encodes an enterocyte brush border disaccharidase, has served as a model for study of intestinal-specific gene expression and differentiation. KGF down-regulated SI mRNA and protein expression in Caco-2 intestinal epithelial cells but not the expression of other brush border enzymes. The down-regulation was dose- and time-dependent and specifically blocked by anti-KGF antibodies. Transfection experiments using SI promoter constructs demonstrated that KGF decreased SI gene transcription. In contrast, the stability of SI mRNA was not affected by incubation of Caco-2 cells with KGF. Electrophoretic mobility shift analysis demonstrated that binding of nuclear proteins to the SI footprint (SIF) 3 and SIF4 regulatory elements within the SI promoter region was increased in Caco-2 cells that had been incubated with KGF. In transfection experiments using a construct in which tandem copies of the SIF4-binding site were inserted upstream of the SV40 promoter and luciferase gene, incubation with KGF resulted in a significant decrease in luciferase activity. However, transfection with a similar construct containing tandem copies of SIF3 had no significant effect on SV40 promoter activity following KGF treatment. SIF4 may bind E4BP4, a previously identified transcriptional repressor protein. This factor may in part mediate the decrease in SI transcription by KGF in Caco-2 cells.

Rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton.

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. These viruses infect mature enterocytes of the small intestine and cause structural and functional damage, including a reduction in disaccharidase activity. It was previously hypothesized that reduced disaccharidase activity resulted from the destruction of rotavirus-infected enterocytes at the villus tips. However, this pathophysiological model cannot explain situations in which low disaccharidase activity is observed when rotavirus-infected intestine exhibits few, if any, histopathologic changes. In a previous study, we demonstrated that the simian rotavirus strain RRV replicated in and was released from human enterocyte-like Caco-2 cells without cell destruction (N. Jourdan, M. Maurice, D. Delautier, A. M. Quero, A. L. Servin, and G. Trugnan, J. Virol. 71:8268-8278, 1997). In the present study, to reinvestigate disaccharidase expression during rotavirus infection, we studied sucrase-isomaltase (SI) in RRV-infected Caco-2 cells. We showed that SI activity and apical expression were specifically and selectively decreased by RRV infection without apparent cell destruction. Using pulse-chase experiments and cell surface biotinylation, we demonstrated that RRV infection did not affect SI biosynthesis, maturation, or stability but induced the blockade of SI transport to the brush border. Using confocal laser scanning microscopy, we showed that RRV infection induces important alterations of the cytoskeleton that correlate with decreased SI apical surface expression. These results lead us to propose an alternate model to explain the pathophysiology associated with rotavirus infection.

Control of gene expression in intestinal epithelial cells.

Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation and adaptation processes in the mammalian intestinal epithelium. Insight into these mechanisms has been gained by the study of transcriptional regulation of the sucrase-isomaltase gene. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes such as sucrase-isomaltase will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.

Sucrase-isomaltase ontogeny: synergism between glucocorticoids and thyroxine reflects increased mRNA and no change in cell migration.

During postnatal maturation of the rat small intestine, glucocorticoid hormones (GC) and thyroxine (T4) act synergistically to elicit a precocious increase of sucrase activity. The current work shows that the synergistic effect on sucrase activity is paralleled by increased steady-state levels of sucrase-isomaltase mRNA. The enhancing effects of T4 on dexamethasone (DEX)-induced sucrase activity was seen even after prolonged treatment (9 days). Moreover, when the location of sucrase-bearing cells was examined after 2 days of hormone treatment, there was distinctly stronger immunostaining of sucrase in the presence of T4, and the sucrase-bearing cells were located on the lower quarter of the intestinal villi regardless of whether the animals received DEX or T4 plus DEX. Thus, despite predictions from the literature, there was no evidence for increased migration in the presence of T4. Instead, we conclude that the synergism between the two hormones is due to greater accumulation of sucrase-isomaltase per epithelial cell.

Premature stimulation of rat sucrase-isomaltase (SI) by exogenous insulin and the analog B-Asp10 is regulated by a receptor-mediated signal triggering SI gene transcription.

The mechanism(s) by which insulin enhance prematurely the activity of brush border membrane (BBM) hydrolases in rat immature intestine is unknown. Therefore, we have compared the responses of four BBM enzymes [sucrase-isomaltase (SI), maltase, lactase-phloridzine hydrolase (LPH), and aminopeptidase] with exogenous insulin, the analog B-Asp10, IGF-I, and antireceptor MAb [insulin-receptor (IR) MAb] given to preweaning pups. Low doses of insulin caused a precocious induction of SI and of SI mRNA and stimulated maltase activity without effect on LPH nor on aminopeptidase activities. IGF-I given at the same dose as that of insulin had no detectable effect on these enzymes. Administration to sucklings of IR MAb prevented the effect of endogenous insulin by inhibiting the expression of SI and maltase without effect on LPH activity. B-Asp10, an insulin analogue that exhibits in vitro a 3.5-fold increase in receptor affinity with sustained signaling of the receptor tyrosine kinase, caused an overexpression of SI by 3.5-fold and of maltase by 1.5-fold compared with equivalent doses of normal insulin. The premature increases in SI activity, SI mRNA, and maltase activity in response to insulin were dose-dependent and were associated with dose-dependent increases in intracellular spermine and spermidine concentrations. In conclusion, these data suggest that the premature induction of SI by insulin is mediated by a dose-dependent signal initiated by binding of the hormone to its intestinal receptor, which after transduction into the cell indirectly triggers the transcription of the SI gene, possibly by changes in intracellular polyamine concentrations.

Suppressive effect of insulin on the synthesis of sucrase-isomaltase complex in small intestinal epithelial cells, and abnormal increase in the complex under diabetic conditions.

An abnormally high level of the sucrase-isomaltase (SI) complex in the small intestine of rats with streptozotocin-induced insulin-dependent diabetes mellitus (IDDM) was normalized in 11 h by the administration of insulin, in addition to normalization of the blood glucose level. Phlorizin, an inhibitor of renal glucose reabsorption, also caused normalization of the blood glucose level in the IDDM rats; however, the level of the SI complex was barely changed. When mucosa explants were cultured in a medium, the SI complex synthesized during the cultivation was accumulated as its precursor protein without maturation, owing to the absence of pancreatic proteases, and the amount of the precursor protein that accumulated in the explants was decreased by the addition of insulin into the medium. Further, the mRNA level of the SI complex in the explants incubated with insulin was obviously lower than that in the absence of insulin. These results indicate that insulin has a suppressive effect on the synthesis of the SI complex, presumably by decreasing the transcriptional level of the gene encoding the complex, in small-intestinal epithelial cells. Thus the synthesis of the SI complex might exceed normal levels in the epithelial cells as a direct result of the depletion of insulin under IDDM conditions.

Research notes: Identification and isolation of chicken sucrase-isomaltase cDNA sequence.

The intestinal sucrase-isomaltase (SI) complex is a glycoprotein of the small intestine brush border membrane that plays an important role in the final degradation of carbohydrate. To clone the chicken SI, we employed reverse transcriptase polymerase chain reaction (RT-PCR). Agarose gel electrophoresis of the PCR products exhibited one amplified band of approximately 800 bp. The fragment was extracted from the gel and sequenced. The cDNA sequence of the chicken SI is 786 bp in length and exhibits 99% identity at the nucleotide level to the Homo sapiens SI mRNA. Using our cDNA as a probe, Northern analysis revealed a transcript of approximately 6.0 kb in chicken jejunum and ileum tissues.

Developmental expression of SI is regulated in transgenic mice by an evolutionarily conserved promoter.

Developmental expression of the sucrase-isomaltase (SI) gene in the mouse intestine involves two major transitions that correspond to critical developmental events. Low levels of SI mRNA were first identified in day 16.5 fetal mouse intestine, immediately after the transition from stratified endoderm to a columnar epithelium organized in nascent villi. Low levels were maintained until the third week of life, when induction of SI mRNA to adult levels was observed coincident with the time of weaning. The mechanism of this pattern of SI gene expression was studied in transgenic mice using a reporter gene construct containing an SI gene promoter that is evolutionarily conserved between mouse and human (nucleotides -201 to +54 of the mouse SI gene). This promoter included the necessary regulatory information to direct transcription to enterocytes in developmental and differentiation-dependent patterns that recapitulated the expression of the endogenous SI gene. However, transgenes lacked the ability to direct induction of precocious expression in suckling animals after administration of corticosteroids. These findings define a short SI gene promoter that contains cis-acting elements that are responsible for developmental and differentiation-dependent transcriptional regulation.