Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are intestinal microvillus membrane hydrolases that play important roles in carbohydrate digestion. These enzymes display characteristic patterns of expression during postnatal development. In rats, lactase specific activity is high at birth and declines during weaning, whereas sucrase specific activity is undetectable at birth and increases to adult levels during weaning (Büller et al. 1990, Henning 1987, Montgomery et al. 1991). These enzymatic changes coincide with the transition from a milk-based diet, in which the primary carbohydrate is lactose, to a diet of solid foods that contain -disaccharides. Biosynthesis of both LPH and SI during postnatal development is regulated primarily by the rate of gene transcription (Krasinski et al. 1994), although posttranscriptional mechanisms for LPH may play a limited role (Freund et al. 1989, Nudell et al. 1993, Sebastio et al. 1989).

In humans, there are two distinct lactase phenotypes: persistent high lactase activity and adult-onset lactase decline (Flatz 1987). In the adult populations with persistent high lactase activity, lactase levels are virtually continuous throughout life. This group is comprised mainly of Caucasians, particularly those descended from Northern and Western Europe (Bayless and Rosensweig 1966, Rao et al. 1994). Adult-onset lactase decline, which is distinct from the rare congenital lactase deficiency characterized by very low or absent lactase levels (Lifschitz 1966, Savilahti et al. 1983), is prevalent in human populations such as Asians and Blacks. This phenotype is characterized by a decline in lactase activity at ages 5-7 years to ~10% of their childhood levels (Büller and Grand 1990, Chung and McGill 1968, Welsh et al. 1978). This pattern is similar to the postweaning decline observed in other mammals, including rats (Büller et al. 1990), rabbits (Sebastio et al. 1989), sheep (Lacey et al. 1994) and pigs (Sangild et al. 1991). Ingestion of high quantities of lactose-containing foods such as milk or other dairy products by individuals with adult-onset lactase decline often results in adverse effects such as bloating, flatulence, distention, abdominal cramps and diarrhea (Bayless et al. 1975, Büller and Grand 1990, Hourigan 1984). The adverse consequences of low intestinal lactase, termed lactose intolerance, often lead to a self-imposed dietary restriction of dairy products that could, in turn, have adverse nutritional effects for the aged. For example, osteoporosis, due to inadequate calcium and vitamin D intake throughout life, has been shown to be associated with individuals with adult-onset lactase decline (Birge et al. 1967, Pacifici et al. 1985).

The effect of old age on disaccharidase activity is poorly understood. In rats, Holt et al. (1991) and Bernard et al. (1992) found that both lactase and sucrase specific activities decrease with aging. In contrast, another study in rats showed that both lactase and sucrase specific activities increase with aging (Raul et al. 1988). LPH and SI mRNA were not measured in any of these studies. In the present investigation, we characterized the relationship between aging and lactase and sucrase activities in the small intestines of adult and senescent rats using a multiple-sampling technique that quantifies total intestinal enzymatic activity. To determine if age-related alterations in disaccharidase activities were associated with changes in the abundance of their mRNA, total intestinal LPH and SI mRNA were also quantified along the length of the rat small intestine. We found that total intestinal lactase and sucrase activities significantly decline with aging in a manner that does not parallel their respective mRNA levels.

MATERIAL AND METHODS

Table 1. Composition of the diet [View Table]

The templates for the antisense LPH and SI mRNA probes have been previously constructed in our laboratory (Krasinski et al. 1994). A template for rat LPH mRNA was constructed from a 117-bp BamHI fragment derived from a previously characterized 1.8-kb rat LPH cDNA (Büller et al. 1990). A previously isolated rat SI cDNA (Traber 1990) served as a template for an antisense RNA probe of mature SI mRNA. This construct contains 271 bases that hybridize to rat SI mRNA. A mouse -actin cDNA (Ambion, Austin, TX) served as a template for a 260-base antisense RNA probe for rat -actin mRNA. The probes were synthesized using a commercially available kit (Riboprobe Gemini II, Promega Biotec). Full-length probes were isolated from denaturing polyacrylamide gels and counted by liquid scintillation counting, and the specific activities were determined. The specific activity was ~200 Bq/fmol for the LPH and SI probes and ~20 Bq/fmol for the -actin probe.

The RNase protection assay was performed as described (Krasinski et al. 1994). RNA samples (5 mg) were hybridized to the probe under high stringency conditions (50% formamide, 68°C) overnight. Unhybridized RNA was digested with RNase A and T1, and the protected fragment was purified and separated in a 6% denaturing polyacrylamide gel. The unhybridized probe was also loaded in a separate lane for size comparison. The gel was dried and exposed to Kodak XAR film (Rochester, NY). The protected fragments were then quantified by excising the bands from the dried gel followed by liquid scintillation counting. Yeast tRNA (10 mg) was analyzed in each experiment to correct for background. RNA from rat liver was used as a negative control for disaccharidase mRNA. All RNA samples were probed at least once with -actin as a control for RNA degradation.

RESULTS

To determine if the pattern of intestinal disaccharidase mRNA changes with aging and to correlate enzymatic activity with mRNA abundance, LPH and SI mRNA were quantified in absolute concentrations using the RNase protection assay. Examples of RNase protection assays demonstrating the proximal-to-distal patterns of mRNA abundance in a 24-mo-old rat, as well as sense strand standards for both LPH and SI, are shown in Figure 2. An RNase protection assay for -actin is also shown as a control for intact and quantified RNA. By comparing the intensity of the protected fragments from sample RNA with that of known amounts of sense strand RNA, absolute concentrations for LPH and SI mRNA were determined. The r-values of the regression lines for all standard curves ranged from 0.95 to 0.99. 

LPH and SI mRNA concentrations in the nine equidistant segments along the small intestines of 3-, 12- and 24-mo-old rats are shown in Figure 3. The horizontal patterns and the mRNA abundance for both disaccharidases did not differ in the three age groups. For all age groups, LPH mRNA was highest in the jejunum and proximal ileum (segments 2-6), and lower in the duodenum (segment 1) and distal ileum (segments 7-9). SI mRNA for all age groups was higher in the duodenum, jejunum and proximal ileum (segments 1-7), with lower levels in the distal ileum (segments 8 and 9).

The horizontal patterns of LPH and SI mRNA abundance can be compared with the respective horizontal patterns of disaccharidase activities. The topographic pattern of LPH mRNA abundance (Fig. 3) was similar to the pattern of lactase activity (Fig. 1). However, the horizontal pattern of SI mRNA abundance (Fig. 3) did not parallel that of sucrase activity (Fig. 1). In all age groups, segments 6 and 7, corresponding to proximal ileum, had abundant SI mRNA concentrations (Fig. 3), but relatively low sucrase activity (Fig. 1).

To determine if changes in total intestinal enzymatic activity and mRNA abundance occur during aging, areas under the proximal-to-distal curves for lactase and sucrase activities and their mRNA were determined (Fig. 4). The actual intestinal length (cm) was used in these calculations. The intestinal length was 98.3 ± 3.7 cm in 3-mo-old rats, 111.0 ± 4.1 cm in 12-mo-old rats and 117.9 ± 7.9 cm in 24-mo-old rats (P = 0.08, ANOVA). As shown in Figure 4, total intestinal lactase and sucrase activities significantly declined with age (P < 0.05). Rats 24 mo of age had mean lactase and sucrase activities that were 12 and 38%, lower, respectively, than those of the 3-mo-old rats (P < 0.05), despite a mean intestinal length that was the highest of all three age groups. In contrast to the age-related decline in enzymatic activities, total intestinal LPH and SI mRNA did not change significantly with aging. Total intestinal lactase and sucrase activities significantly declined with aging in a manner that does not parallel the abundance of their respective mRNA.

DISCUSSION

Although intestinal disaccharidase activities decline significantly with age, this decrease was not paralleled by a decline in LPH or SI mRNA. The decrease in lactase and sucrase activities is therefore likely due to a diminished efficiency in mRNA translation, an increase in protein degradation, and/or a partial inactivation of the disaccharidase active site, rather than a reduced transcription rate. Age-related declines of both protein synthesis and degradation have been described in several organs including liver, kidney, heart and brain (Goldspink et al. 1985). In rats, Holt et al. (1991) found that overall mucosal proteins in jejunum and ileum do not change with age, but both sucrase activity and SI protein decrease. These data suggest that specific microvillus membrane proteins may decline with age and that this decrease is likely due to a decline in enzyme protein rather than an inactivation of the disaccharidase activity. Studies in pancreatectomized rats (Alpers and Tedesco 1975) and in a mouse model of exocrine pancreatic insufficiency (Kwong et al. 1978) showed that the presence of pancreatic proteases may result in lower disaccharidase activity. However, there is no evidence that aging is associated with an increase in pancreatic proteases (Greenberg and Holt 1986).

An interesting observation that could explain the age-related decrease in intestinal disaccharidase activities is suggested by changes in the distribution of SI along the crypt-villus axis. In the proximal intestine of adult rats, immunohistochemistry revealed that SI is present on villus cells beginning at the crypt-villus junction, and extending up the entire villus, whereas in senescent animals, SI immunoreactivity begins several cells above the crypt-villus junction (Holt and Kotler 1987, Holt et al. 1985). This results in an increased proportion of less differentiated epithelial cells (i.e., those not producing SI) at the base of the villi. Although cell migration rates (Holt et al. 1983), villus cell number and villus heights (Holt et al. 1984) do not change with aging, older rats have an increased number of crypt cells (Holt et al. 1984), crypt cell proliferation rates (Holt and Yeh 1989) and zone of proliferation within the crypt (Holt et al. 1988) compared with younger animals. An increase in the crypt cell population could result in a delay in cellular differentiation along the crypt-villus axis that is manifested by less differentiated cells at the base of the villi in senescent animals. The cumulative effect of a reduced number of villus enterocytes synthesizing SI could result in a lower total intestinal sucrase activity in the aged rats. The same mechanism might also explain a lower lactase activity in aged animals.

For all age groups, the pattern of SI mRNA abundance along the small intestine was different than that of sucrase activity. Specifically, SI mRNA was discordant in segments 6 and 7 in which SI mRNA abundance was high and sucrase activity was low. In the ileum of adult rats, less SI mRNA was associated with polyribosomes than in the jejunum suggesting that the translational efficiency is decreased in the distal intestine (Hoffman and Chang 1991 and 1992). Another possible explanation is the effect of pancreatic proteases on sucrase activity. Using monoclonal antibodies specific for the sucrase or isomaltase activity in enzyme-linked immunosorbent assays, Goda et al (1988) found higher amounts of isomaltase immunoreactivity compared with that of sucrase in rat jejunum. This was explained by a selective degradation effect by pancreatic proteases on sucrase activity. Further study is required to characterize the posttranscriptional events that might result in a lower sucrase activity in the distal intestine.

At present it is not clear whether the age-related declines in lactase and sucrase activities observed in rats can be extrapolated to humans. Although studies in humans indicate that aging is not significantly correlated with a decline in disaccharidase activity (Cuatrecasas et al. 1965, Wallis et al. 1993, Welsh et al. 1978), one study revealed that lactase, sucrase and maltase specific activities were lowest in the group of subjects over 70 y of age compared with all other adult (>20 y) age-decade groups (Welsh et al. 1978). It is noteworthy, however, that a limited number of people were examined in all of these studies, making inferences about age-related alterations in disaccharidase activity speculative.

It is also not clear whether the magnitude of the declines in lactase and sucrase activities has physiological importance if extrapolated to humans. Although a 12% decline in lactase activity probably is of little consequence, of greater concern is a 38% decrease in sucrase activity. LPH is required for the digestion of lactose in milk and is thus crucial for the nutrition of neonates. Sucrase, however, digests -disaccharides, which are present in solid foods that occur in the diets of adult and senescent individuals. Thus, an age-related decline in sucrase activity could have implications for the digestion of carbohydrate contained in the diet of the aged. Additional studies are therefore required to determine if there is a relationship between aging and disaccharidase activity in humans.

ACKNOWLEDGMENT