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Molecular Nutrition, Kagawa Nutrition University, Sakado, Saitama 350-02, Japan and * Biochemistry, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo, Tokyo 113, Japan
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENT
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Lactase activity declines during postnatal development in rats, but little is known about the underlying molecular mechanism of this phenomenon. We attempted to clarify whether the regulation was at the transcriptional or post-transcriptional level and to examine the effects of dietary factors on that regulation. Newborn rats were divided into two groups, prolonged nursing and weaning, at d 21. The prolonged nursing rats were nursed for a further 6 d, whereas weaning rats were separated from their dams and fed nonpurified diet for the same period. The patterns of declining lactase protein and mRNA concentrations during weaning were determined by Western blot and Northern blot analyses, respectively, and compared with lactase activity. There were significant (P < 0.001) correlations between them: r = 0.97 for specific activity vs. protein, r = 0.99 for specific activity vs. mRNA and r = 0.96 for protein vs. mRNA. The lactase activity per milligram DNA showed a pattern similar to that of the specific activity. This result argues against the decline in lactase activity being due to the dilution caused by newly synthesized materials during the weaning period and suggests transcriptional regulation. Furthermore, the prolonged nursing rats showed the same results as weanlings for lactase protein, mRNA, specific activity and activity per milligram DNA. These observations indicate that the regulation of lactase expression is at the transcriptional level and that it is not affected by the termination of milk ingestion.
KEY WORDS: rats · lactase · Northern blot analysis · Western blot analysis · weaningINTRODUCTION
Lactase-phlorizin hydrolase (EC 3.2.1.23-62, subsequently referred to as lactase) is an enzyme indispensable for all mammalian neonates including humans. The structural characteristics of the lactase gene and the amino acid sequence of the lactase protein have been described by Mantei et al. (1988)
for humans and rabbits, and by Duluc et al. (1991) for rats. However, the molecular basis for the processing of its precursor molecule into a mature membrane-bound form and the regulation of its expression 
both during development and along the length of the small intestine has been poorly understood and remains to be investigated (Van Beers et al. 1995). The specific activity is elevated during suckling, declines markedly during weaning and remains thereafter at a level corresponding to 20% of that at birth (Henning 1981). Transcriptional regulation during development was suggested in rats (Büller et al. 1990, Krasinski et al. 1994), humans (Fajardo et al. 1994) and sheep (Lacey et al. 1994) on the basis of the parallelism among levels of lactase activity, lactase mRNA and immunoreactive lactase protein concentrations. Conversely, some studies suggested post-transcriptional regulation (Freund et al. 1991, Nudell et al. 1993). More recently, Troelsen et al. (1992) identified a trans-acting nuclear factor, designated NF-LPH1, from pig intestine that binds to the regulatory cis-element CE-LPH1 in the lactase promoter region. They showed that NF-LPH1 is intestine specific and more abundant in infant pigs than in adult pigs.
During the weaning period, all mammals change their source of nutrition from milk to a nonmilk diet. Therefore, it is reasonable to suppose that dietary lactose in milk may affect lactase expression. In adult rats, Goda et al. (1995) induced lactase expression by diet manipulation, feeding the rats a high starch diet. Duluc et al. (1992) analyzed the distribution of lactase expression along the length of intestine and were able to modify this pattern by changing the luminal content in preweaned rats but not in adult rats. However, little is known about the effect of lactose on lactase expression in rats at the suckling-weaning transition except for the preliminary works of Henning (1981) and Lebenthal et al. (1973) whose results showing no appreciable effects. To examine the role of diet, especially that of milk, on lactase expression in greater depth, rats were subjected to prolonged nursing, and lactase activity, lactase protein and mRNA were determined.
MATERIALS AND METHODS
and Labarca and Paigen (1980), respectively. Lactase activity was measured as described by Dahlqvist (1968) and expressed on the basis of milligrams protein or milligrams DNA. For the purification of poly(A)+RNA from total cellular RNA, tissue was processed immediately after decapitation, and total cellular RNA and poly(A)+RNA were isolated with the commercial kits, Wako Isogen (Wako Pure Chemical, Osaka, Japan) and Oligotex-dT30 (Takara Shuzo, Kyoto, Japan), respectively, in accordance with the manufacturers' manuals.
cDNA.
cDNA encoding rat lactase was synthesized using a reverse transcriptase and a polymerase chain reaction as follows. We synthesized two primers, 5
-CTGCCAAGCTTCACTGAGGA-3 (primer 1) and 5-GTACCAAGCTTCATGCCATTGTTGGCAATGA-3 (primer 2), corresponding to nucleotides 3574-3594 and the inverse complement of nucleotides 4091-4110 of the published rat lactase cDNA sequence (Duluc et al, 1991) with the addition of a five-base+Hind III site. For the strand conversion of RNA into DNA, 0.3 µg of poly(A)+RNA obtained from 5-d-old rat proximal intestine was incubated with 5 units of reverse transcriptase (Takara Shuzo) and 0.75 µg of primer 2 in the mixture, according to the manufacturer's instructions (final volume, 30 µL). After incubation at 42°C for 1 h, the enzyme was denatured by heating the mixture at 95°C for 5 min. A standard polymerase chain reaction was performed by using a GeneAmp DNA amplification reagent kit from Takara Shuzo as follows: the two primers and 3 µL of the above reaction mixture containing synthesized cDNA as a template were added to a 30-µL mixture. After the reaction, a single discrete 525-bp species was identified by 4% agarose gel electrophoresis. The amplified DNA, recovered from the gel by Gene Clean II (BIO 101, Vista, CA), was digested with Hind III and cloned into pBluescript M13+ vector. It was confirmed that the previously known sequence (Duluc et al. 1991) was also included in this clone. A cDNA clone for rat 
-actin was kindly provided by H. Hamada of the Tokyo Metropolitan Institute of Medical Science.
Western blot analysis.
For the preparation of lactase antibody, lactase was purified from the proximal intestine by a published method (Skovbjerg et al. 1981). The purification factor was ~700 and revealed a single protein band of 125-kDa molecular weight by SDS-PAGE. Polyclonal antibody was prepared by injecting a rabbit and was used for the Western blot analysis. An aliquot of the homogenate of mucosal cells, containing 30 µg of protein, was analyzed by SDS-PAGE on a 3% stacking gel and a 7.5% separating gel (Laemmli 1970). Six SDS-PAGE gels were run, two for the d 27 litters (27 P and 27 W), one for each of the d 8, d 21 and adult groups. The first five gels contained six lanes as well as a size marker. The six lanes with two d-27 gels each consisted of three P samples and three W samples. The six lanes in the d 8, d 21 and adult gels consisted of one sample per rat in each group. The sixth gel consisted of five pooled samples, one for each group of six rats, i.e., d 8, d 21, 27 P, 27 W and adult. Proteins were separated and transferred to a polyvinylidene difluoride fluorotrans membrane (Japan Genetics, Tokyo, Japan) using the Trans-Blot Cell apparatus (Bio-Rad, Richmond, CA). For the immunoreaction, this membrane was incubated with polyclonal antibody as described previously by Sakuma et al. (1996). Briefly, after the membrane was washed, immunogenic lactase bands were identified using a second antibody conjugated to alkaline phosphatase followed by development of the enzyme reaction with 4-chloronaphthol. The lactase bands were visualized as a colored product, and densities were quantified with a Shimadzu scanning densitometer CS-9000 (Kyoto, Japan). The means of the six lanes from each litter on a membrane were calculated and compared statistically. No statistical analyses were performed on the pooled samples.
Northern blot analysis.
In the present work, -actin was chosen as a marker for standardization of the amount of mRNA applied to each gel well. Denatured poly(A)+RNA (5 µg) was electrophoresed through 0.8% agarose with 2.2 mol/L formaldehyde, then blotted to a Hybond-N nylon membrane (Amersham, Arlington Heights, IL), exactly as described by Sakuma et al. (1987). Six agarose gels were run in exactly the same format as described above. Ribosomal RNA were used as size markers on an additional lane. Hybridization was conducted at 65°C overnight in a solution of 0.5 mol/L sodium phosphate buffer, pH 7.2, 7% SDS and 1 mmol/L EDTA containing a labeled probe of lactase. The amount of 32P radioactivity was ~0.17 MBq. As a probe for the hybridization, the rat lactase cDNA and rat -actin cDNA were labeled by the random primer hexamer method with (
-32P)dCTP (Amersham International, Amersham, UK) using a commercial kit, Random Primer DNA Labeling Kit (Takara Shuzo) in accordance with the manufacturer's manual. Hybridization was detected by autoradiography with Fuji X-ray film (Fuji Shashin Film, Tokyo, Japan) at 
80°C. This same membrane was also hybridized with a -actin probe in an identical fashion as the lactase probe. Before repeat hybridization, the membrane was treated with 0.1% SDS at 95°C with autoradiographic verification of removal of the first probe. The values of lactase and -actin mRNA in each lane were determined by densitometry as described above, and the final amount of lactase message was then corrected for -actin. The means of the six samples from each litter on an autoradiograph were calculated and tested statistically.
Statistical analysis.
All results were expressed as means ± SEM. We tested the daily difference in mean values of body weight and mucosal cell weight between the P and W groups with Student's t test for unpaired samples. Growth rate was compared between the two groups, combining all values from d 21 to 27 by one-way ANOVA. The difference between the P and the W groups at d 27 was tested with Student's t test for unpaired samples. One-way ANOVA with a Bonferroni test (Keppel 1991) was used for comparison during development concerning specific activity, mRNA and protein concentrations. Coefficients of correlation between activity per milligram protein and DNA were calculated using Microsoft Excel Version 4.0 (Microsoft, Tokyo, Japan). Coefficients of correlation between specific activity, mRNA and protein concentrations and statistical differences among them were calculated by the same method. Differences were considered significant at P < 0.05.
RESULTS
), care was taken as described above in Materials and Methods to obtain similar growth rates for the P and W groups. There was no significant difference in either body weight or daily change of body weight between the P and W groups during the period from d 21 to 27. However, in spite of precautions taken, a difference in growth rates for the two groups began to develop around d 30, probably caused by malnutrition. Therefore we decided to terminate the prolonged nursing at d 27 to eliminate the effects of possible malnutrition.
DISCUSSION ACKNOWLEDGMENT 
Fig. 2.
Lactase activity of control and experimental rat litters during development by two determinations: activity per milligram protein (panels A and B) and per milligram DNA (panel C), and comparison between the prolonged nursing (P) and weaning (W) rats at d 27. The homogenate of total proximal intestinal mucosal cells was determined for activity, and data are shown as the amount of liberated glucose per milligram protein and per milligram DNA. In panel A, six rats from each of 10 control litters were killed at each date indicated. Six 15-wk-old rats were used as adult rats. Data are expressed as means ± SEM, n = 6. There was a significant correlation (P < 0.001, r = 0.81) between activity per milligram protein and per milligram DNA. There was no significant difference between the P and W rats at d 27 in lactase activity, either per milligram protein or per milligram DNA.
[View Larger Version of this Image (21K GIF file)]
Fig. 3.
Western blot analysis of lactase protein from the experimental P (prolonged nursing) and W (weaning) rat groups at d 27 (panel A); the same results compared with those of the d 8 and 21 control litters and adults are shown in panel B. The homogenate of total proximal intestinal mucosal cells containing 30 µg protein was analyzed by SDS-PAGE for each of six rats in a group and visualized by lactase antibody after transferring to membrane. Panel A shows the results of only one of the experimental litters at d 27. The arrow indicates the position of the lactase protein (125 kDa). The rats from the other experimental litter at d 27 and all rats of the d 8, d 21 and adult groups were individually analyzed in the same manner (data not shown). The lactase bands of five membranes were quantified by densitometer and mean values for each litter were calculated. Statistical analysis is presented in Figure 5 In each lane in panel B, 30 µg protein from the pooled homogenate of each of the d 8, d 21, 27 P, 27 W and adult groups was analyzed. The STD lane in both panels indicates the locations of molecular weight standards. Pooled samples were not statistically analyzed.
[View Larger Version of this Image (40K GIF file)]
-actin mRNA, respectively, for one experimental litter at d 27, i.e., the three 27 P and the three 27 W rats. Each of the rats from the other experimental litter and the d 8, d 21 and adult groups were examined in the same way (data not shown). The panel shows clearly that there was no difference between the 27 P and 27 W samples. This was later confirmed by statistical analysis.
Fig. 4.
Northern blot analysis of lactase mRNA from the experimental P (prolonged nursing) and W (weaning) rat groups at d 27 (panels A and B); panels C and D show the same results in comparison with those of the d 8 and 21 control litters and adults. Total RNA was extracted from proximal intestinal mucosal cells and mRNA was further isolated. This mRNA fraction (5 µg) was analyzed on an agarose gel (one for each six rats of a group), transferred to a membrane and hybridized with (-32P)dCTP-labeled lactase cDNA. Panel A shows the result from only one experimental litter of three 27 P and three 27 W rats as an autoradiograph. Thereafter, all radioactivity was washed out from the membrane and rehybridization was conducted for -actin mRNA, shown in panel B. The rats from the other experimental litter at d 27 and all rats of the d 8, d 21 and adult groups were individually analyzed in the same manner (data not shown). All lactase bands on autoradiographs were quantified by densitometer and mean values for each litter were calculated. Statistical analysis is presented in Figure 5. In panel C, a 5-µg sample from the pooled mRNA of each of the d 8, d 21, 27 P and 27 W groups was hybridized with lactase cDNA and rehybridized with -actin cDNA (panel D). The arrows indicate the locations of ribosomal 18S RNA and 28S RNA. Pooled samples were not statistically analyzed.
[View Larger Version of this Image (75K GIF file)]
-actin mRNA, respectively, with the pooled samples of a group in the order of d 8, d 21, 27 P, 27 W and adult rats. This result was consistent with the patterns of specific activity (Fig. 2) and Western blot analysis (Fig. 3, panel B).
Fig. 5.
Lactase specific activity (panel A), lactase protein (panel B) and mRNA (panel C) of control and experimental rat litters during development. Data from d 8, d 21 and adult rats are shown in comparison with P (prolonged nursing) and W (weaning) rats at d 27. All data are calculated as a percentage of the values of d 8 and expressed as means ± SEM, n = 6. Means sharing a common superscript letter in each panel are not significantly different at P < 0.05. There was no significant difference between 27 P and 27 W in lactase specific activity, lactase protein and mRNA. Significant (P < 0.001) correlations were as follows: r = 0.97 for activity vs. protein, r = 0.99 for activity vs. mRNA and r = 0.96 for protein vs. mRNA.
[View Larger Version of this Image (22K GIF file)]
). Previously, Cousineau and Green (1980) purified lactase from proximal and distal regions of intestine, with different molecular moieties. Accordingly, all experiments and determinations in this study were conducted only with the proximal intestine.
), and the composition of milk revealed a specific pattern during the course of lactation (Keen et al. 1981). In pups, an increase in the plasma concentration of thyroxine and corticosterone parallels the decline in lactase activity occurring at weaning (Henning 1981), and experimental changes by hormone injection (Freund et al. 1990, Yeh et al. 1991) or starvation (Freund et al. 1991) modulate lactase activity. On the basis of these observations, post-transcriptional and pre-translational regulation have been suggested by Freund et al. (1990 and 1991), whereas Yeh et al. (1991) suggested post-translational regulation. Meanwhile, in the promoter sequences of the lactase gene, no potential binding sites for receptors of the above hormones were found (Boll et al. 1991). Accordingly, it has been suggested that these hormones do not exert a direct effect on lactase gene transcription.
, Sakuma et al. 1996), an apparent decrease in the extent of the decline of lactase expression was observed, with rather higher activity in prolonged nursing rats compared with that of weanlings. Consequently, some effect from dietary factors on lactase expression at weaning has been suggested by these investigators. However, we consider that such results from stunted pups must be confounded to some extent by differential growth rates. Therefore, to eliminate weight loss of the dams as a factor, litter size was decreased to six and the time schedule of prolonged nursing was conducted as described in Materials and Methods. As shown in Figure 1, there was no significant difference in the growth rate between the prolonged nursing rats and weanlings. Thus, in contrast to previous studies, including our own, we were able to produce results free of the effect of differential growth rates.
) and the developmental change of the activity of intestinal glycosylating enzymes (Lenoir et al. 1995). However, in our study, immunoreactive lactase protein levels determined by Western blot analysis correlated well with lactase activity. Therefore, regulation at the transcriptional level seems more likely than translational or post-translational regulation. Furthermore, on the Western blot pattern, the values of the prolonged nursing rats decreased to a low level similar to that of the weanlings at d 27; thus the dietary manipulation of prolonged nursing had no effect.
, Krasinski et al. 1994) and in sheep by Lacey et al. (1994). However, there have been conflicting suggestions concerning developing rats. Nudell et al. (1993) found no correlation between lactase protein and mRNA and suggested translational or post-translational regulation. Recently, the promoter region of the lactase gene was functionally analyzed (Boukamel et al. 1994, Troelsen et al. 1992) and the hypothesis of transcriptional regulation has been strongly supported. Southwestern blot analysis conducted with nuclear extracts prepared from the tissues of suckling rats or pigs indicated that a cis-element, CE-LPH1, interacted specifically with a nuclear protein, NF-LPH1 (Boukamel et al. 1994). Furthermore NF-LPH1 was shown to be important in identifying the tissue specificity of lactase expression and its decline at weaning, because the amount of NF-LPH1 is in direct proportion to lactase activity (Troelsen et al. 1992). We conclude that transcriptional regulation is crucial in defining the developmental decline of lactase expression at weaning; however, more than a single mechanism must be involved. Although each aspect of weaning must be physiologically related via various intermediaries with the dietary transition from milk to a nonmilk diet, it is unlikely that the termination of milk ingestion acts as a cue for the decline of lactase expression in rats.
FOOTNOTES
Manuscript received 4 November 1996. Initial reviews completed 12 December 1996. Revision accepted 6 May 1997.
LITERATURE CITED
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