Lactase-phlorizin Hydrolase mRNA Research Articles

The possible roles of homeobox protein, Cdx-2 for the expression of LPH gene during postnatal development

The expression of intestinal lactase-phlorizin hydrolase (LPH) gene normally decreases after completion of weaning in almost all mammals. To elucidate the mechanism whereby LPH gene expression is regulated during the suckling–weaning period, we studied the effects of the thyroid hormone (T 3) on LPH gene expression in the small intestine during postnatal development in the rat. Firstly, we measured LPH mRNA level in rat jejunum at 5, 13, 20 and 27 days after birth. The amount of LPH mRNA at 27 days was significantly lower than that at 5 days. The transcript level of Cdx-2, which is a putative transcriptional factor for regulation of LPH gene expression, was also significantly decreased after 21 days. The binding of nuclear protein to the cis element CE-LPH1 on the promoter region of the LPH gene was reduced at the end of the weaning period. Daily intraperitoneal (i.p.) injection of T 3 for 6 days during days 22–27 significantly reduced LPH mRNA level by day 27 (50%, P < 0.01), but injection of T 3 during days 8–13 did not. Moreover, i.p. T 3 injection during days 22–27 was accompanied by a reduction in the level of Cdx-2 mRNA. Our study suggests that the decrease in the LPH gene expression during the weaning period is associated with a reduction of Cdx-2 expression caused by thyroid hormone.

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.

Hepatocyte nuclear factor-1α is required for expression but dispensable for histone acetylation of the lactase-phlorizin hydrolase gene in vivo

Hepatocyte nuclear factor-1alpha (HNF-1alpha) is a modified homeodomain-containing transcription factor that has been implicated in the regulation of intestinal genes. To define the importance and underlying mechanism of HNF-1alpha for the regulation of intestinal gene expression in vivo, we analyzed the expression of the intestinal differentiation markers and putative HNF-1alpha targets lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) in hnf1alpha null mice. We found that in adult jejunum, LPH mRNA in hnf1alpha(-/-) mice was reduced 95% compared with wild-type controls (P < 0.01, n = 4), whereas SI mRNA was virtually identical to that in wild-type mice. Furthermore, SI mRNA abundance was unchanged in the absence of HNF-1alpha along the length of the adult mouse small intestine as well as in newborn jejunum. We found that HNF-1alpha occupies the promoters of both the LPH and SI genes in vivo. However, in contrast to liver and pancreas, where HNF-1alpha regulates target genes by recruitment of histone acetyl transferase activity to the promoter, the histone acetylation state of the LPH and SI promoters was not affected by the presence or absence of HNF-1alpha. Finally, we showed that a subset of hypothesized intestinal target genes is regulated by HNF-1alpha in vivo and that this regulation occurs in a defined tissue-specific and developmental context. These data indicate that HNF-1alpha is an activator of a subset of intestinal genes and induces these genes through an alternative mechanism in which it is dispensable for chromatin remodeling.

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4.

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.

Glucagon-like peptide 2 enhances maltase-glucoamylase and sucrase-isomaltase gene expression and activity in parenterally fed premature neonatal piglets.

Exogenous glucagon-like peptide 2 (GLP-2) mimics the stimulatory effect of enteral nutrition on intestinal mucosal growth in preterm neonatal pigs. Little is known about its effects on small intestinal function. In this study, we investigated whether the trophic actions of GLP-2 and enteral nutrition are paralleled by effects on small intestinal function. Cesarean-delivered piglets (92% of gestation) were given either a parenteral nutrient infusion [total parenteral nutrition (TPN), n = 7], TPN + human GLP-2 (25 nmol/kg/d, n = 8), or enteral nutrition (ENT, n = 6) for 6 d. Gene expression (mRNA) and activities of lactase phlorizin hydrolase (LPH), maltase-glucoamylase (MGA), sucrase-isomaltase (SI), aminopeptidase N (ApN), and A (ApA) and dipeptidyl peptidase IV (DPP IV) were measured. Both GLP-2 and enteral nutrition increased mucosal weight (+30-40%, p < 0.05) relative to TPN. GLP-2 stimulated jejunal MGA and SI mRNA abundance and activity levels but did not change LPH in parenterally fed pigs (p < 0.05). Enteral nutrition decreased jejunal LPH and MGA mRNA abundance and activity and increased ileal ApN, ApA, and DPP IV activities relative to TPN (p < 0.05). We conclude that GLP-2 and enteral nutrition exert different effects on intestinal enzyme function despite similar effects on intestinal growth. In addition, the effects of GLP-2 on intestinal function in these parenterally fed, premature neonatal pigs differed from those previously reported for similarly fed term neonates.

Glucagon-Like Peptide 2 Enhances Maltase-Glucoamylase and Sucrase-Isomaltase Gene Expression and Activity in Parenterally Fed Premature Neonatal Piglets

Exogenous glucagon-like peptide 2 (GLP-2) mimics the stimulatory effect of enteral nutrition on intestinal mucosal growth in preterm neonatal pigs. Little is known about its effects on small intestinal function. In this study, we investigated whether the trophic actions of GLP-2 and enteral nutrition are paralleled by effects on small intestinal function. Cesarean-delivered piglets (92% of gestation) were given either a parenteral nutrient infusion [total parenteral nutrition (TPN), n = 7], TPN + human GLP-2 (25 nmol/kg/d, n = 8), or enteral nutrition (ENT, n = 6) for 6 d. Gene expression (mRNA) and activities of lactase phlorizin hydrolase (LPH), maltase-glucoamylase (MGA), sucrase-isomaltase (SI), aminopeptidase N (ApN), and A (ApA) and dipeptidyl peptidase IV (DPP IV) were measured. Both GLP-2 and enteral nutrition increased mucosal weight (+30–40%, p < 0.05) relative to TPN. GLP-2 stimulated jejunal MGA and SI mRNA abundance and activity levels but did not change LPH in parenterally fed pigs (p < 0.05). Enteral nutrition decreased jejunal LPH and MGA mRNA abundance and activity and increased ileal ApN, ApA, and DPP IV activities relative to TPN (p < 0.05). We conclude that GLP-2 and enteral nutrition exert different effects on intestinal enzyme function despite similar effects on intestinal growth. In addition, the effects of GLP-2 on intestinal function in these parenterally fed, premature neonatal pigs differed from those previously reported for similarly fed term neonates.

Intestinal protein and LPH synthesis in parenterally fed piglets receiving partial enteral nutrition and enteral insulinlike growth factor 1.

Providing partial enteral nutrition (PEN) supplemented with insulinlike growth factor-1 (IGF-1) to parenterally fed piglets increases lactase-phlorizin hydrolase (LPH) activity, but not LPH mRNA. The current aim was to investigate potential mechanisms by which IGF-1 up-regulates LPH activity. Newborn piglets (n = 15) received 100% parenteral nutrition (TPN), 80% parenteral nutrition + 20% parenteral nutrition (PEN), or PEN + IGF-1 (1.0 mg. kg-1. d-1) for 7 days. On day 7, [2H3]-leucine was intravenously administered to measure mucosal protein and brush border LPH (BB LPH) synthesis. Weight gain, nutrient intake, and jejunal weight and length were similar among the treatment groups. Partial enteral nutrition alone increased mucosal weight, villus width and cross-sectional area, LPH activity, mRNA expression, and high mannose LPH precursor (proLPHh) abundance compared with TPN (P<0.05). Insulinlike growth factor-1 further increased mucosal weight, LPH activity, and LPH activity per unit BB LPH approximately twofold over PEN alone (P < 0.05) but did not affect LPH mRNA or the abundance of proLPHh (one of the LPH isoforms) or mature LPH. Isotopic enrichment of [2H3]-leucine in plasma, mucosal protein, and LPH precursors, and the fractional and absolute synthesis rates of mucosal protein and LPH were similar among the treatment groups. Insulinlike growth factor-1 treatment increased total mucosal protein synthesis (60%, P < 0.05) but not LPH synthesis compared with the other two groups. Because IGF-1 did not affect the fractional synthesis rate of either mucosal protein or LPH, the authors suggest that enteral IGF-1 increases mucosal protein mass and LPH activity by suppressing mucosal proteolytic degradation.

Intestinal Protein and LPH Synthesis in Parenterally Fed Piglets Receiving Partial Enteral Nutrition and Enteral Insulinlike Growth Factor 1

ABSTRACTBackgroundProviding partial enteral nutrition (PEN) supplemented with insulinlike growth factor‐1 (IGF‐1) to parenterally fed piglets increases lactase–phlorizin hydrolase (LPH) activity, but not LPH mRNA. The current aim was to investigate potential mechanisms by which IGF‐1 up‐regulates LPH activity.MethodsNewborn piglets (n = 15) received 100% parenteral nutrition (TPN), 80% parenteral nutrition + 20% parenteral nutrition (PEN), or PEN + IGF‐1 (1.0 mg · kg−1 · d−1) for 7 days. On day 7, [2H3]‐leucine was intravenously administered to measure mucosal protein and brush border LPH (BB LPH) synthesis.ResultsWeight gain, nutrient intake, and jejunal weight and length were similar among the treatment groups. Partial enteral nutrition alone increased mucosal weight, villus width and cross‐sectional area, LPH activity, mRNA expression, and high mannose LPH precursor (proLPHh) abundance compared with TPN (P &lt;0.05). Insulinlike growth factor‐1 further increased mucosal weight, LPH activity, and LPH activity per unit BB LPH approximately twofold over PEN alone (P &lt; 0.05) but did not affect LPH mRNA or the abundance of proLPHh (one of the LPH isoforms) or mature LPH. Isotopic enrichment of [2H3]‐leucine in plasma, mucosal protein, and LPH precursors, and the fractional and absolute synthesis rates of mucosal protein and LPH were similar among the treatment groups. Insulinlike growth factor‐1 treatment increased total mucosal protein synthesis (60%, P &lt; 0.05) but not LPH synthesis compared with the other two groups.ConclusionsBecause IGF‐1 did not affect the fractional synthesis rate of either mucosal protein or LPH, the authors suggest that enteral IGF‐1 increases mucosal protein mass and LPH activity by suppressing mucosal proteolytic degradation.

Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet.

The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [(13)C(1)]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha, but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.

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.

Investigation of Three Doses of Oral Insulin-like Growth Factor-I on Jejunal Lactase Phlorizin Hydrolase Activity and Gene Expression and Enterocyte Proliferation and Migration in Piglets

In a previous study, oral IGF-I at 65 nM increased lactase phlorizin hydrolase (LPH) activity and villus height in piglets, however, the mechanisms were unknown. Herein, the response to a range of doses of IGF-I was investigated and we hypothesized that LPH and villus height would respond to oral IGF-I in a dose-dependent manner. Two 14-d experiments were conducted using cesarean-derived piglets. In experiment 1, piglets (n = 28) were fed formula containing 0, 33, 65, or 131 nmol/L (0, 0.25, 0.5, or 1.0 mg/L) recombinant human IGF-I. In experiment 2, 5'-bromodeoxyuridine was administered to piglets fed formula alone (n = 4) or containing 131 nmol/L IGF-I (n = 4). IGF-I did not affect body weight gain or intestinal weight or length. Jejunal villus height and LPH activity were significantly greater in piglets fed 131 nmol IGF-I/L than control piglets. Villus height and lactase activity in piglets fed the 33 and 65 nmol/L IGF-I doses were similar and intermediate between control and 131 nmol IGF-I/L. Jejunal mRNA expression and LPH polypeptide abundance were investigated in piglets receiving 0 or 131 nmol/L IGF-I. Steady state LPH mRNA abundance was significantly higher (p < 0.05) in IGF-I-treated piglets. The relative abundance of proLPH(h) was not significantly increased (p = 0.06) by IGF-I treatment. Mucosal DNA content and DNA synthesis were greater in piglets receiving 131 nmol IGF-I/L than control, however, enterocyte migration and mucosal protein content were unaffected. Thus, oral IGF-I increased jejunal LPH activity and LPH mRNA abundance and stimulated intestinal cell hyperplasia in normal piglets.

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.

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.

Dietary carbohydrates enhance lactase/phlorizin hydrolase gene expression at a transcription level in rat jejunum.

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.

GATA-6 stimulates a cell line-specific activation element in the human lactase promoter

Lactase-phlorizin hydrolase (LPH) synthesis is restricted to differentiated small intestinal enterocytes and is highly regulated during development. Analysis of expression of LPH promoter segments fused with luciferase transfected in Caco-2 cells, a line that uniquely expresses LPH mRNA, mapped an 18-base pair (bp) segment 100 bp upstream of the transcription start site that is required for transactivation. Remarkably, the LPH upstream element (LUE) has no stimulatory activity in both human intestinal and nonintestinal lines in which LPH mRNA is absent. Electrophoretic analysis of sequence-specific DNA-nuclear protein complexes demonstrated the presence of a Caco-2 cell-specific protein(s) (CCP), which is uniformly absent in LPH nonproducer cell lines. Mutational analysis of the LUE demonstrated that bases contained within a GATA consensus motif are critical for both CCP binding and transcription from the LPH promoter. Caco-2 cells express high levels of GATA-6 mRNA in a cell line-specific manner, suggesting that GATA-6 is a CCP that complexes with the LUE. When expressed by a plasmid, GATA-6 transactivated the LPH promoter. The stimulation was abrogated with mutations in the GATA consensus motif as well as mutations in a flanking downstream element. These studies are consistent with an important role of an intestinal GATA binding protein in cell type-specific transactivation of the LPH promoter.

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.

Lactase Phlorizin Hydrolase Synthesis Is Decreased In Protein-Malnourished Pigs

We have examined the effect of protein malnutrition on brush border (BB) lactase phlorizin hydrolase (LPH) synthesis in young pigs. Two groups of four 3-wk-old pigs were fed diets containing either 19 g soy protein, 63 g carbohydrate and 5 g fat per 100 g diet (a protein-sufficient diet) or 3 g soy protein, 85 g carbohydrate and 5 g fat per 100 g diet (a protein-deficient diet). After 8 wk of consuming the diets, pigs were infused intravenously with 2H3-leucine for 8 h, then killed. The jejunum was collected for measurement of lactase activity, LPH mRNA abundance and the rate of LPH post-translational synthesis. Lactase activities did not differ between groups (mean 8.1 ± 1.2 μmol·min−1·g mucosa−1). LPH mRNA abundance relative to elongation factor-1α mRNA (the constitutive/reference mRNA) was significantly (P < 0.05) higher in well-nourished pigs (0.36 ± 0.03%) than in protein-malnourished pigs (0.21 ± 0.02%). The rate constants of BB LPH post-translational synthesis were also significantly higher in the well-nourished (103 ± 9%·d−1) than in the protein-malnourished pigs (66 ± 8%·d−1). Further, the absolute synthesis rate of BB LPH, a measure of the amount of enzyme synthesized per gram of tissue, was significantly higher in well-nourished than in protein-malnourished pigs (in arbitrary units, 892 ± 90 vs. 450 ± 34, respectively). Thus, protein malnutrition affects both LPH mRNA abundance and post-translational processing in young pigs.

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.

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.

Heterogeneity of Intestinal Lactase Activity in Children: Relationship to Lactase-Phlorizin Hydrolase Messenger RNA Abundance

Despite extensive study in both humans and nonhuman mammals the mechanisms which regulate intestinal lactase activity, particularly during development, are incompletely understood. Our previous studies of human adults are consistent with an important role of lactase-phlorizin hydrolase (LPH) mRNA abundance in determining the lactase persistence/nonpersistence phenotypes. Our intent in the present study was to determine the role of LPH mRNA in the regulation of lactase in children. We therefore studied duodenal mucosal biopsies from 39 children undergoing diagnostic upper endoscopy in whom significant small intestinal and nutritional disease was excluded. We found no relationship between the level of LPH mRNA and lactase enzymatic activity. Our observations suggest the importance of posttranscriptional mechanisms in lactase regulation in human children.