Expression of lactase-phlorizin hydrolase in sheep is regulated at the RNA level.
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ABSTRACT:
SUBMITTER: Lacey SW
PROVIDER: S-EPMC1137319 | biostudies-other | 1994 Sep
REPOSITORIES: biostudies-other
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Project description:We have previously shown that dietary sucrose stimulates the lactase/phlorizin hydrolase (LPH) mRNA accumulation along with a rise in lactase activity in rat jejunum [Goda, Yasutake, Suzuki, Takase and Koldovský (1995) Am. J. Physiol. 268, G1066-G1073]. To elucidate the mechanisms whereby dietary carbohydrates enhance the LPH mRNA expression, 7-week-old rats that had been fed a low-carbohydrate diet (5.5% of energy as starch) were given diets containing various monosaccharides or sucrose for 12h. Among carbohydrates examined, fructose, sucrose, galactose and glycerol elicited an increase in LPH mRNA accumulation along with a rise in lactase activity in the jejunum. By contrast, glucose and alpha-methylglucoside were unable to elicit a significant increase in LPH mRNA levels. To explore a transcriptional mechanism for the carbohydrate-induced increases in LPH mRNA levels, we employed two techniques currently available to estimate transcriptional rate, i.e. RNA protection assays of pre-mRNA using an intron probe, and nuclear run-on assays. Both assays revealed that fructose elicited an increase in transcription of the LPH gene, and that the transcription of LPH was influenced only slightly, if at all, by glucose intake. These results suggest that certain monosaccharides such as fructose or their metabolite(s) are capable of enhancing LPH mRNA levels in the small intestine, and that transcriptional control might play a major role in the carbohydrate-induced increase of LPH mRNA expression.
| S-EPMC1219342 | biostudies-other
Project description:Background and aimsThe mechanism of the developmental downregulation of the lactase-phlorizin hydrolase (LPH) gene underlying adult-type hypolactasia is unknown. We have determined the functional significance of the recently identified two single nucleotide polymorphisms (SNPs), C/T(-13910) and G/A(-22018), associated with adult-type hypolactasia by studying LPH mRNA levels in intestinal biopsy samples with different genotypes.MethodsIntestinal biopsy samples were taken from 52 patients with abdominal complaints. Hypolactasia was diagnosed by determining lactase and sucrase activities and calculating their ratio (L/S ratio). The functional effect of the C/T(-13910) and G/A(-22018) genotype on expression of LPH mRNA was demonstrated in patients heterozygous for the C/T(-13910) and G/A(-22018) polymorphism and an informative expressed SNP located in the coding region of the LPH mRNA. Reverse transcription-polymerase chain reaction followed by solid phase minisequencing was used for accessing the relative expression levels of the LPH alleles using informative SNPs located in exons 1, 2, 6, 10, 13, or 17 as markers.ResultsStatistically significant differences between the three different genotypes CC(-13910) GG(-22018), CT(-13910) GA(-22018), and TT(-13910) AA(-22018) and their respective L/S ratios were observed. Relative quantitation of the expressed LPH alleles showed that the persistent allele represented 92 (6)% (mean (SEM), range 78-99%; n=14) of the expressed LPH mRNA. The patient with the homozygous persistent TT(-13910) AA(-22018), as well as hypolactasic patients with CC(-13910) GG(-22018), showed equal expression of both alleles (47 (1)%; n=7).ConclusionsExpression of LPH mRNA in the intestinal mucosa in individuals with T(-13910) A(-22018) alleles is several times higher than that found in individuals with C(-13910), G(-22018) alleles. These findings suggest that the two SNPs, C/T(-13910) and G/A(-22018), associated with adult-type hypolactasia, are associated with the transcriptional regulation of the LPH gene. The presence of the T(-13910) A(-22018) allele also shows significant elevation of the L/S ratio.
| S-EPMC1773659 | biostudies-literature
Project description:The longitudinal expression of two brush-border enzymes, lactase-phlorizin hydrolase (EC 3.2.1.23/62) and aminopeptidase N (EC 3.4.11.2), was studied in the small intestine of the post-weaned pig. Whereas the level of mRNA, encoding aminopeptidase N (relative to that of beta-actin), only varied moderately from the duodenum to the terminal ileum, the amount of lactase-phlorizin hydrolase mRNA exhibited a sharp maximum in the proximal jejunum. For both enzymes, the level of protein synthesis, studied in cultured mucosal explants, correlated well with the level of mRNA, and no major variation in post-translational processing or intracellular transport was observed along the intestine. The mRNA/specific-activity ratio for both enzymes was markedly (3-5-fold) higher in the duodenum and proximal jejunum, compared with the ileum. This indicates an increased proximal turnover rate, most likely caused by the presence in the gut lumen of pancreatic proteases. In neonatal animals, the level of mRNA for lactase-phlorizin hydrolase in both proximal and distal regions of the intestine was of the same magnitude as in the proximal jejunum of the post-weaned pigs. Our results point to two mechanisms that affect the expression of lactase-phlorizin hydrolase in the pig during development: (1) a primary regulation at the level of mRNA (predominantly in the ileum); (2) an increased rate of turnover of the enzyme, mainly in the duodenum and proximal jejunum, and most likely due to an increased secretion into the gut lumen of pancreatic proteases (a mechanism also affecting aminopeptidase N and probably other brush-border enzymes as well).
| S-EPMC1134835 | biostudies-other
Project description:The maturational decline in lactase-phlorizin hydrolase (LPH) activity was studied in groups of young rats ranging from suckling to early post-weaned states. Associated maturational increases in sucrase-isomaltase (SI) and maltase-glucoamylase (MG) activities were also examined as a comparison. Over this time period changes in cellular concentrations of the three enzymes were observed, reflecting corresponding changes in enzyme activities. Synthesis patterns accompanying these maturational changes in concentration were examined using labelled leucine as a marker. Synthesis of LPH was found to be maintained at constant rates independent of the maturation-associated decline in its concentration, whereas the increases in cellular concentrations of SI and MG were due to accelerated synthesis of the enzyme. Turnover of LPH, based on both the fractional synthesis rate and the disappearance rate of labelled leucine from prelabelled LPH pools, was increased in a quantitatively similar way to the decline in LPH concentration. These findings are consistent with our earlier proposal that the maturational decline of LPH occurs because of accelerated turnover, without a decrease in its rate of synthesis.
| S-EPMC1130896 | biostudies-other
Project description:The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2 kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3 kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator.Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22.In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28 days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7 days and declines significantly by 28 days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells.Thus, a 3.3-kb LPH 5' flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.
| S-EPMC3408868 | biostudies-literature
Project description:Lactase-phlorizin hydrolase is exclusively expressed in the small intestine and is often used as a marker for the differentiation of enterocytes. The cis-element CE-LPH1 found in the lactase-phlorizin hydrolase promoter has previously been shown to bind an intestinal-specific nuclear factor. By electrophoretic mobility-shift assay it was shown that the factor Cdx-2 (a homoeodomain-protein related to caudal) binds to a TTTAC sequence in the CE-LPH1. Furthermore it was demonstrated that Cdx-2 is able to activate reporter gene transcription by binding to CE-LPH1. A mutation in CE-LPH1, which does not affect Cdx-2 binding, results in a higher transcriptional activity, indicating that the CE-LPH1 site contains other binding site(s) in addition to the Cdx-2-binding site.
| S-EPMC1218263 | biostudies-other
Project description:Previous research has found that milk is associated with a decreased risk of colorectal cancer (CRC). However, it is unclear whether the milk digestion by the enzyme lactase-phlorizin hydrolase (LPH) plays a role in CRC susceptibility. Our study aims to investigate the direct causal relationship of CRC risk with LPH levels by applying a two-sample Mendelian Randomization (MR) strategy. Genetic instruments for LPH were derived from the Fenland Study, and CRC-associated summary statistics for these instruments were extracted from the FinnGen Study, PLCO Atlas Project, and Pan-UK Biobank. Primary MR analyses focused on a cis-variant (rs4988235) for LPH levels, with results integrated via meta-analysis. MR analyses using all variants were also undertaken. This analytical approach was further extended to assess CRC subtypes (colon and rectal). Meta-analysis across the three datasets illustrated an inverse association between genetically predicted LPH levels and CRC risk (OR: 0.92 [95% CI, 0.89-0.95]). Subtype analyses revealed associations of elevated LPH levels with reduced risks for both colon (OR: 0.92 [95% CI, 0.89-0.96]) and rectal cancer (OR: 0.92 [95% CI, 0.87, 0.98]). Consistency was observed across varied analytical methods and datasets. Further exploration is warranted to unveil the underlying mechanisms and validate LPH's potential role in CRC prevention.
| S-EPMC10975724 | biostudies-literature
Project description:Lactase-phlorizin hydrolase is a brush-border enzyme which is specifically expressed in the small intestine where it hydrolyses lactose, the main carbohydrate found in milk. We have previously demonstrated in transgenic mice that the tissue-specific and developmental expression of lactase is controlled by a 1 kb upstream region of the pig lactase gene. Two homeodomain transcription factors, caudal-related homeodomain protein (Cdx2) and hepatic nuclear factor 1alpha (HNF1alpha), are known to bind to regulatory cis elements in the promoters for several intestine-specific genes, including lactase, and are present in mammalian intestinal epithelia from an early stage in development. In the present study, we examined whether Cdx2 and HNF1alpha physically interact and co-operatively activate transcription from the lactase-phlorizin hydrolase promoter. We show that the presence of both factors leads to a much higher level of transcription than the sum of the activation by either factor alone. The N-terminal activation domain of Cdx2 is required for maximal synergy with HNF1alpha. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1alpha. The interaction domain includes the homeodomain region of both proteins. This is the first demonstration of a functional interaction between two transcription factors involved in the activation of a number of intestine-specific genes. Synergistic interaction between tissue-restricted factors is likely to be an important mechanism for reinforcing developmental and tissue-specific gene expression within the intestine.
| S-EPMC1220882 | biostudies-other
Project description:The biosynthesis and maturation of the human intestinal lactase-phlorizin hydrolase (LPH; EC 3.2.1.23-3.2.1.62) has been studied in cultured intestinal biopsies and mucosal explants. Short time pulse labelling revealed on high mannose intermediate of Mr 215,000 which was converted upon endo-beta-N-acetylglucosaminidase H (endo-H) digestion to a polypeptide of Mr 200,000. The brush border form of LPH was revealed after longer pulse periods and has Mr 160,000. It possesses mainly complex oligosaccharide chains and, owing to its partial endo-H sensitivity, at least one chain of the high mannose type. Leupeptin partially inhibited the appearance of the Mr-160,000 polypeptide. Monensin treatment of biopsies resulted in the modification of the Mr-160,000 species to the Mr-140,000 molecule, which was endo-H sensitive. Pulse-chase analysis indicated a slow post-translational processing of the high mannose precursor (Mr 215,000) to yield the mature brush-border form (Mr 160,000) of LPH. Our results further indicate that LPH is synthesized as a single polypeptide precursor which is intracellularly cleaved to yield the mature brush border of LPH. The data presented suggest that this cleavage occurs during the translocation of the molecule across the Golgi complex.
| S-EPMC1147578 | biostudies-other
Project description:Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.
| S-EPMC1133415 | biostudies-other