Lactase-phlorizin Hydrolase Expression Research Articles

Lactose Intolerance: What Your Breath Can Tell You.

Lactose intolerance has seen recognized as a clinical syndrome characterized by pain, abdominal distention, flatulence, and diarrhea after the consumption of lactose. Lactose is a common disaccharide found in dairy that requires lactase-phlorizin hydrolase (LPH) to break down into glucose and galactose. A deficiency in this enzyme results in flora bacteria further along in the gastrointestinal tract (GI) tract to metabolize the excess lactose to numerous gases, including H2. Recent studies show that the risk of symptoms after lactose ingestion depends on the dose of lactose, LPH expression, intestinal flora, and sensitivity of the gastrointestinal tract. Currently, there are several diagnostic tests that investigate the biological mechanism of lactose intolerance such as blood, biopsy, genetic, and breath tests. Due to its relatively low cost, availability, and non-invasiveness, the hydrogen breath test (HBT) has become a popular technology to aid in the diagnosis of many gastroenterological diseases, specifically lactose intolerance. Additionally, while administering the HBT there seems to be a lack of uniform criteria amongst the various studies, with many using their own guidelines, which may in turn cause inconsistency with the analysis of the results. With ever improving nanotechnology, novel approaches to expedite and lower the costs of the HBT has become an area of research with significant advancements.

Intestinal microbiota differentially affect brush border enzyme activity and gene expression in the neonatal gnotobiotic pig

To study microbial influence on intestinal development pertaining to nutrient digestion, two separate gnotobiotic experiments were performed, each with 16 piglets allocated to four treatment groups: germfree (GF), monoassociation with Escherichia coli, monoassociation with Lactobacillus fermentum or conventionalization with faecal bacteria (CV). Enzyme activity and gene expression of lactase phlorizin hydrolase (LPH) and aminopeptidase N (APN) were measured in isolated enterocytes, harvested on day 14, using specific substrates and quantitative PCR respectively. Enterocytes of CV pigs had reduced APN activity, but had increased gene expression relative to GF, making the specific activity:mRNA (A:G) ratio dramatically lower (p < 0.05). Similarly, LPH A:G ratio was significantly reduced (p < 0.05) in enterocytes of CV pigs as compared with GF. The results of co-incubation of L. fermentum, E. coli and faecal bacteria with APN indicate a direct relationship between enzyme inactivation and specific A:G ratio in enterocytes. We conclude that enterocyte up-regulation of APN expression occurs as either a direct response to microbial colonization or as a feedback mechanism in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host with the intestinal microbiota for available nutrients.

Higher expression of jejunal LPH gene in rats fed the high-carbohydrate/low-fat diet compared with those fed the low-carbohydrate/high-fat diet is associated with in vitro binding of Cdx-2 in nuclear proteins to its promoter regions

It has been previously demonstrated that the expression of lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) genes are higher in rats fed a high-carbohydrate/low-fat (HCT) diet than in those fed a low-carbohydrate/high-fat (LCT) diet. In the present study, using a nuclear run-on assay we clearly show that higher expression of LPH and SI genes in jejunum of rats fed the HCT diet compared with those fed a LCT diet was regulated at the transcription levels. DNase I foot printing analysis of the 5' flanking region of the rat LPH gene demonstrated that by incubating the jejunal nuclear extract the protected region was conserved as the same sequence as the homeodomain protein-binding element designated as CE-LPH1. UV-cross linking and electromobility shift assay in vitro clearly showed that Cdx-2 was including proteins bound to CE-LPH1. Moreover, in vitro binding of Cdx-2 to CE-LPH1 as well as SIF1, a cis-element identified as the binding element of Cdx-2 on the SI gene, in jejunal nuclear extracts of rats fed a HCT diet were greater than those fed a LCT diet. These results suggest that in vitro binding of Cdx-2 to CE-LPH1 as well as SIF1 in jejunal nuclear extracts is associated with the higher expression of the LPH and SI genes in rats fed the HCT diet compared with those fed a LCT diet.

Lactose and Lactase—Who Is Lactose Intolerant and Why?

Lactase-phlorizin hydrolase (LPH) is expressed only in the small intestine and is confined to absorptive enterocytes on the villi with a tightly controlled pattern of expression along the proximal to distal and crypt-villus axes of the intestine. LPH expression is regulated mainly at the level of lactase (LCT) gene transcription that directs 2 phenotypes: a decline in LCT activity (LCT nonpersistence) in mid-childhood in the majority of the world's population, and maintenance of the lactase levels found in infancy (LCT persistence) in people of northern European extraction and scattered populations elsewhere. The molecular mechanisms that regulate these phenotypes are not completely understood. A population genetic association of lactase persistence with 2 single nucleotide polymorphisms in the distal 5'-flanking region of LCT (-13.9T and -22A) has been confirmed in northern Europeans, but this fails to explain lactase persistence found in some African groups. Any hypothesis for the control of lactase expression must reconcile the presence of high levels of activity in early life in all humans and the characteristic loss of activity found subsequently in many but not all people.

T −13910 DNA variant associated with lactase persistence interacts with Oct-1 and stimulates lactase promoter activity in vitro

Two phenotypes exist in the human population with regard to expression of lactase in adults. Lactase non-persistence (adult-type hypolactasia and lactose intolerance) is characterized by a decline in the expression of lactase-phlorizin hydrolase (LPH) after weaning. In contrast, lactase-persistent individuals have a high LPH throughout their lifespan. Lactase persistence and non-persistence are associated with a T/C polymorphism at position -13,910 upstream the lactase gene. A nuclear factor binds more strongly to the T-13,910 variant associated with lactase persistence than the C-13,910 variant associated with lactase non-persistence. Oct-1 and glyceraldehyde-3-phosphate dehydrogenase were co-purified by DNA affinity purification using the sequence of the T-13,910 variant. Supershift analyses show that Oct-1 binds directly to the T-13,910 variant, and we suggest that GAPDH is co-purified due to interactions with Oct-1. Expression of Oct-1 stimulates reporter gene expression from the T and the C-13,910 variant/LPH promoter constructs only when it is co-expressed with HNF1alpha. Binding sites for other intestinal transcription factors (GATA-6, HNF4alpha, Fox and Cdx-2) were identified in the region of the -13,910 T/C polymorphism. Three of these sites are required for the enhancer activity of the -13,910 region. The data suggest that the binding of Oct-1 to the T-13,910 variant directs increased lactase promoter activity and this might provide an explanation for the lactase persistence phenotype in the human population.

Regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression.

Lactase-phlorizin hydrolase (LPH) is an enterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along the crypt-villus axis and along the length of the small bowel. Previous studies from the authors' laboratory have demonstrated that 2 kb of the 5'-flanking region of the rat LPH gene control the correct tissue, cell, and crypt-villus expression in transgenic animals. To examine further the regulation conferred by this region, protein-DNA interactions were studied using DNase I footprint analyses in LPH-expressing and nonexpressing cell lines. Functional delineation of this 5'-flanking sequence was performed using deletion analysis in transient transfection assays. Studies revealed a generally positive activity between -74 and -37 bp, a cell-specific negative region between -210 and -95 bp, and additional elements further toward the 5'-terminus that conferred a highly cell-specific response in reporter activity. Computer analysis of distal regions encompassing identified footprints revealed potential binding sites for various intestinal transcription factors. Co-transfection and electromobility shift assay experiments indicated binding of HNF3beta at three sites relevant to LPH expression. The data demonstrate that the cell specificity of LPH gene expression depends upon both positive and negative interactions among elements in the first 2 kb of the LPH 5'-flanking region.

The Specific Expression Patterns of Lactase, Sucrase and Calbindin-D9k in Weaning Rats Are Regulated at the Transcriptional Level

During weaning, rat lactase-phlorizin hydrolase (LPH) expression decreased to the low levels found in adults, while sucrase-isomaltase (SI) sharply increased. Calbindin-D9k (CaBP) is specific to the intestine and expression peaked within a few days of weaning. The present study investigates whether these molecules are regulated at transcriptional or post-transcriptional levels and examines the effects of diet on regulation. At normal weaning on day 21, litters were separated from their dams and one group was fed with a standard laboratory diet (weaned (W) group). The other group received a diet containing lactose as the sole source of carbohydrate (lactose-fed (L) group). Mucosal cells were obtained from the proximal part of the rat small intestine and then the activity and concentration of LPH, SI and CaBP proteins and mRNAs were determined. Three parameters revealed the same changing patterns in LPH, SI and CaBP during development and there was significant (p<0.001) correlation between three parameters: LPH, r=0.97 for activity vs. protein, r=0.99 for activity vs. mRNA, r=0.96 for protein vs. mRNA, SI, r=0.99 for activity vs. protein, r=0.98 for activity vs. mRNA, r=0.96 for protein vs. mRNA, CaBP, r=0.94 for activity vs. protein, r=0.97 for activity vs. mRNA, r=0.95 for protein vs. mRNA. Expression of the three proteins did not differ between the L and W groups. Accordingly, it has been suggested that the expression of LPH, SI and CaBP during development is defined at the transcriptional level and dietary changes do not exert a primary effect on it.

An enhancer activates the pig lactase phlorizin hydrolase promoter in intestinal cells

Lactase phlorizin hydrolase is a small intestinal-specific brush border protein commonly used as a specific marker of differentiated enterocytes. A number of transcription factors involved in the enterocyte-specific expression of lactase phlorizin hydrolase have been identified. An upstream regulatory region, which we have named the "LPH enhancer", located at position -894 to -798 in the porcine lactase phlorizin hydrolase gene, is necessary for high differentiation-dependent LPH expression in intestinal cells. The LPH enhancer was studied by mutation analysis, transfection experiments and electrophoretical mobility shift assays. The LPH enhancer is active in intestinal cells (Caco-2) and not in non-intestinal cells (HeLa). The LPH enhancer is only able to enhance expression when it is located in front of an intestinal-specific promoter such as the lactase phlorizin hydrolase promoter or the sucrase-isomaltase promoter. In front of an SV40-derived promoter the LPH enhancer has no stimulatory effect. In addition to the lack of promoter-promiscuity, the LPH enhancer is not a classical enhancer in the sense that it is not orientation-independent and it cannot function when located 3' of a reporter gene. The LPH enhancer contains at least three cis-elements (at -894 to -880, -880 to -875 and -833 to -814) with functional importance for the LPH enhancer activity.

Defective Intracellular Processing of Lactase‐Phlorizin Hydrolase Protein in Rats Prenatally Exposed to Ethanol

We have previously shown that fetal exposure to ethanol in rats produces both structural and biochemical abnormalities in absorptive enterocytes. Among the indicators of injury are derangements in the expression of lactase-phlorizin hydrolase (LPH), which is an essential enzyme for the assimilation of milk. In an animal model of fetal alcohol syndrome, unsuckled newborn rats prenatally exposed to maternal ethanol revealed a 10- to 15-fold increase in the number of LPH mRNA molecules per absorptive enterocyte, compared with controls (Estrada et al., Alcohol. Clin. Exp. Res. 20:1662-1668, 1996). However, lactase activity per cell was similar in both groups. The aim of this study was to characterize the effect of prenatal exposure to ethanol on the processing of LPH mRNA and protein. RNase protection assays using 3'- and 5'-directed antisense RNA probes revealed that the LPH mRNA from ethanol-exposed pups is full length. However, metabolic labeling, followed by immunoprecipitation using an anti-LPH monoclonal antibody, demonstrated a significant alteration in LPH protein processing. Intestinal explants from 21-day ethanol-exposed fetuses that were chased 30 min after a [35S]methionine pulse showed greater amounts of newly synthesized LPH precursors (205 and 220 kDa) and low molecular weight degradation products than controls. However, despite the increases in LPH precursor, the amount of 130 kDa mature LPH was similar in ethanol-exposed and control explants. These data suggest an increase in intracellular degradation of LPH precursor in rats prenatally exposed to ethanol, which occurs before its insertion into the microvillus membrane. Biosynthesis of LPH appears to be upregulated at the transcriptional level, which overcomes the degradation of LPH precursor during processing.

A possible role of a nuclear factor NF-LPH1 in the regional expression of lactase-phlorizin hydrolase along the small intestine.

Lactase-phlorizin hydrolase (LPH), an enterocyte-specific disaccharidase, displays not only a post weaning decline but also regional differences in the small intestine. To investigate the mechanisms of regional LPH expression along the small intestine, the correlation between LPH mRNA abundance, lactase activity and the amount of a nuclear factor (NF-LPH1) binding to a cis-element was determined in various intestinal segments of suckling and adult rats. In suckling rats, both LPH mRNA and lactase activity were expressed at maximum in the jejunum, but they were hardly detected in the colon. In adult rats, both LPH mRNA and lactase activity were the highest in the jejunum and virtually absent in the ileum. Lactase activity and LPH mRNA abundance in suckling rats were 2-3 times more than those of adult rats in all regions of the small intestine. An electromobility shift assays of nuclear proteins revealed that NF-LPH1 was present in rat small intestine as well as in Caco-2 cells. The amount of NF-LPH1 binding to the cis-element was also approximately 2-fold more in the intestinal nuclear extracts of suckling rats than that of adult rats. NF-LPH1 was detected in all regions of the small intestine in both suckling and adult rats. In both cases, the amounts of NF-LPH1 binding to the cis-element increased from the duodenum to upper jejunum, and decreased toward the ileum. The coordinate postnatal declines of LPH mRNA and NF-LPH1 expression in various regions of the small intestine suggest that NF-LPH1 might be involved not only in the regulation of postnatal LPH gene expression but in region-specific LPH gene expression as well.

Regulation of sucrase and lactase in Caco-2 cells: relationship to nuclear factors SIF-1 and NF-LPH-1.

Recent studies suggest the importance of two transcription factors, Cdx-2 and NF-LPH-1, in the regulation of sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH) gene expression, respectively. Cdx-2 accounts for the tissue specificity of sucrase expression (16), and NF-LPH-1 varies with postnatal changes in lactase activity, suggesting a role in its developmental regulation (22). We used electrophoretic mobility shift assays to study the relationship of Cdx-2 and NF-LPH-1 to SI and LPH gene expression in Caco-2 cells to provide evidence regarding the role of these factors in the development of sucrase and lactase with cellular differentiation. We found that Cdx-2 levels correlated with SI expression and that NF-LPH-1 did not correlate with LPH expression. These studies suggest a role for Cdx-2 but not for NF-LPH-1 in the development of carbohydrase expression in these cells.

Expression of lactase-phlorizin hydrolase in sheep is regulated at the RNA level.

Lactase-phlorizin hydrolase (LPH) is expressed on the intestinal brush border and is responsible for the hydrolysis of lactose, the chief sugar in mammalian milk. The enzyme activity of LPH peaks soon after birth in most mammals and declines to much lower levels before adolescence. The molecular basis of this pattern of expression has not been clearly established. We have measured relative amounts of LPH mRNA in intestine from sheep with ages across a developmental spectrum, including third trimester fetal lambs, newborn lambs and adult sheep. LPH mRNA levels in the jejunum decline approximately 50-fold between infancy and adulthood, in parallel with the reduction in both lactase specific activity and immunologically reactive lactase protein expression in sheep jejunum. LPH mRNA is present in high concentration in the duodenum of newborn lambs, but steadily declines by day 34 and is dramatically reduced in adults. Because the changes in LPH mRNA, protein, and enzymic activity are generally parallel, we conclude that the developmental regulation of LPH in sheep is probably mediated primarily at the mRNA level.