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Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762-5759 and * Membrane Transport Research Group, Department of Physiology, B.P. 6128, Succursale Centre-ville, Université de Montréal, Montréal, Québec, Canada H3C3J7
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
The interplay between suckling, intestinal growth and brush-border membrane functions is critical during the perinatal period. The present study investigates changes in intestinal dimensions, activities of four brush border membrane hydrolases (lactase, sucrase, maltase and aminooligopeptidase) and rates of sugar and amino acid uptake by intact tissues and brush border membrane vesicles during the first 24 h of suckling. Total intestinal weight, mucosal weight and protein content increased 58%, 80% and 126% (P < 0.05) during the first 6 h of suckling; length and surface area did not increase. Total mucosal DNA content was 4.6-fold higher at 24 h after birth, with the rate of increase differing among intestinal regions. Hydrolytic capacities of the entire small intestine increased, more so for homogenates than for brush border membrane vesicles, and more for lactase relative to the other hydrolases studied. Rates of nutrient transport declined, especially for brush border membrane vesicles, for proximal and mid-intestine relative to distal intestine, and for glucose relative to galactose and amino acids. We conclude that 1) changes in brush border membrane digestive functions coincide with rapid intestinal growth, with postnatal patterns varying among hydrolases, transporters and regions; 2) insertion into the brush border membrane, not synthesis, limits the postnatal increase of hydrolase activity; and 3) despite declines in specific activity, hydrolytic and glucose transport capacities of the entire intestine remained stable or increased, and exceeded estimated dietary loads because of intestinal growth.
Key words: colostrum, neonatal, pigs, nutrient transport, brush border hydrolases.INTRODUCTION
The interaction of diet, intestinal growth and digestive functions is critical during the perinatal period when mammals switch from placental to enteral nutrition. The onset of suckling triggers rapid postnatal intestinal growth in several species [e.g., rats (Berseth et al. 1983
), rabbits (Gall and Chung 1982) and dogs (Schwarz and Heird 1994)], but the lack of intestinal growth in kittens during the first week after birth (Buddington and Diamond 1992) indicates the response is not universal. In pigs, the rapid postnatal intestinal growth elicited by colostrum (Widdowson et al. 1976) has been attributed to endocytosis of ingested immunoglobulins, mucosal hyperplasia and protein synthesis (Burrin et al. 1992, Simmen et al. 1990). It is accompanied by changes in intestinal morphology (Xu et al. 1992) and enterocyte ultrastructure (Komuves et al. 1993).
Much less is known about the consequences of rapid postnatal intestinal growth on intestinal functions. The best known example is for rats at weaning, when the rapid proliferation of enterocytes coincides with the appearance of sucrase activity and fructose transport (Henning 1987, Toloza and Diamond 1992). Even though lactase specific activity declines during this period, total intestinal lactase activity remains relatively stable because of intestinal growth (Montgomery et al. 1991). Several changes also accompany the rapid perinatal intestinal growth in pigs. These include declines in rates of monosaccharide transport (Puchal and Buddington 1992), shifts in the processing of lactase gene products (Burrin et al. 1994), changes in brush border membrane (BBM)4 physical and chemical properties (Alessandri et al. 1990), and the loss of endocytic functions (Westrom et al. 1989).
The present study defines the effect of feeding during the first 24 h after birth when the pig intestine undergoes dramatic changes in structure and functions. This was accomplished by correlating intestinal growth with functional properties. A related objective was to understand the effect of intestinal growth on the functional capacities of the entire length of small intestine. This was accomplished by measuring intestinal dimensions, protein and DNA content, rates of nutrient transport, and the activities of four BBM hydrolases [lactase, sucrase, maltase and aminooligopeptidase (AOP)]. Because measurements were made at three sites along the entire length of small intestine at four ages between birth and 24 h, our results provide better resolution of spatial and temporal changes and the associated correlations than in previous studies that evaluated either fewer or more distant time points and in most cases only one region of the intestine. We provide much needed data about changes in nutrient uptake during the neonatal period, and whereas there are numerous studies of the disaccharidases, relatively little is known about AOP and other peptidases, despite their importance in protein digestion. Pigs were chosen as the model because of the relevance for studying digestion in humans (Moughan et al. 1992).
MATERIALS AND METHODS
70°C.
Measurements of intestinal dimensions.
From the middle of each region we removed a 10-cm segment. After recording of wet weight, each segment was slit along the length for measurement of circumference, which was used to calculate nominal surface area (without accounting for area amplification by villi and microvilli). These values were used to determine regional weights and surface areas, which were summed to calculate values for the entire intestine. The percentage of mucosa was based on dry weight after gently scraping each segment with a glass slide and drying the mucosa and underlying tissues to a constant weight (48 h, 45-50°C). Mucosal percentages were used to calculate regional and total quantities of mucosa.
Measurements of brush border membrane hydrolase activities.
One of the two aliquots of frozen mucosa from each region of each pig was used to prepare brush border membrane vesicles (BBMV) by CaCl2 precipitation (Schmitz et al. 1973), which is reported to yield higher enzyme recovery than MgCl2 (Ibrahim and Balasubramanian 1995). The BBMV were suspended in 20 mmol/L Tris-HEPES-100 mmol/L mannitol buffer (pH 7.5), and protein content of homogenates and BBMV was determined by the Coomassie Blue method (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as the standard.
; AOP (EC 3.4.11.2) activity was assayed by the method of Wojnarowska and Gray (1975) using 0.17 mmol/L leucyl-
-naphthylamide. In addition, we measured lactase and sucrase activities in the different fractions that resulted during preparation of BBMV using mucosa from the three regions of pigs suckled for 6 h (n = 4). Enzyme activities [micromoles of substrate hydrolyzed per minute (IU)] were normalized to protein content. Total
intestinal hydrolase activities (millimoles hydrolyzed per minute) were calculated by summing the hydrolytic capacities of each region (product of BBMV/homogenate specific activity in each region × BBMV/homogenate protein content per gram mucosa × regional mucosal weight).
) was used to quantify DNA content. Aliquots of mucosal homogenates were added to 33258 Hoechst fluorochrome (Sigma Chemical, St. Louis, MO), which was dissolved in water and had a final concentration of 1.5 µmol/L. Fluorescent readings were recorded using excitation and emission wavelengths of 360 and 450 nm, respectively, and compared with a standard prepared with calf thymus DNA.
Measurements of initial rates of brush border membrane vesicle hexose and amino acid uptake.
Studies of BBMV transport followed the same methods used to define glucose and amino acid transport by fetal and neonatal pigs (Buddington and Malo 1996). Specifically, BBMV were prepared from the second frozen aliquots of mucosa by MgCl2 precipitation and suspended in 50 mmol/L Tris-HEPES buffer (pH 7.5) with 0.1 mmol/L MgSO4, 200 mmol/L KCl and 125 mmol/L mannitol. Aliquots (25 µL, 10-40 g/L) were stored in liquid nitrogen until used for transport measurements (within 48 h of final BBMV preparation). Initial rates of uptake were measured at 25°C using a rapid filtration technique and a fast sampling, rapid filtration apparatus. On the basis of our previous studies (Buddington and Malo 1996), a total of nine time points were used over 4.5 s for glucose and 20 s for the amino acids. Tracer levels of 3H-labeled D-glucose and L-amino acids and liquid scintillation counting were used to quantify accumulation of glucose and amino acids by BBMV. Final concentrations in the incubation media were 50 mmol/L Tris-HEPES buffer (pH 7.5) with 0.1 mmol/L MgSO4, 96 mmol/L NaCl, 104 mmol/L KCl, 125 mmol/L D-mannitol with 4 µmol/L [3H]D-aldohexose or 2 µmol/L [3H]L-amino acid.
Characterizing kinetics of Na+/D-glucose cotransport using intact tissues.
Rates of uptake by 0.51-cm2 intact tissues from each region were measured as described previously (Puchal and Buddington 1992). Briefly, pieces of tissues were secured by silk ligatures onto the tips of 0.5-cm stainless steel rods with the mucosa exposed. Beginning 45 min after death, the tissues were preincubated for 5 min in aerated, 37°C mammalian Ringer's solution. They were then exposed for 2 min to 37°C Ringer's solution with 0.1, 1, 5, 10, 25 and 50 mmol/L D-glucose with tracer levels of 14C-labeled D-glucose and 3H-labeled L-glucose. Each solution was aerated and stirred by a bar rotating at 1200 rpm to reduce unstirred layer influences and maintain tissue oxygenation. The tissues were rinsed for 20 s in cold Ringer's solution, removed from the rods and placed in tared vials; wet weight was recorded. Accumulation of radioactive D- and L-glucose by the tissues was measured using liquid scintillation counting. Rates of uptake were calculated and normalized to tissue wet weight; rates of uptake based on tissue surface area yielded similar conclusions. Maximum total intestinal glucose transport capacities (millimoles per minute) were calculated as the sum of the regional transport capacities (product of rates of transport at 50 mmol/L in each region times regional wet weight).
Statistics.
Data presented in tables and figures are means ± SEM. We used two-way ANOVA (SAS 1992) to detect effects of age and region on measured variables, with P < 0.05 accepted as the critical value. When a significant effect was detected, Duncan's multiple range test (Zar 1974) was used to identify specific differences between ages and/or regions. In the figures, we indicate significant differences between 6, 12 and 24 h suckled pigs in comparison with 0 h pigs, but in the tables we also indicate where differences were detected among the four age groups. Regional comparisons are described in the text of the results.
).
RESULTS
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Table 1. Quantitative features of the small intestines of pigs suckled by sows for 0, 6, 12 and 24 h after birth1 |
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Table 2. Small intestinal mucosal protein and DNA content of pigs suckled by sows for 0, 6, 12 and 24 h after birth1 |
DISCUSSION 
Fig. 3.
Total intestinal maltase activity (units, panel A) and specific activity [µmol/(min·mg protein), panel B] for homogenates and brush border membrane vesicles (BBMV) prepared from the proximal (circles), mid (squares) and distal (triangles) small intestines of pigs, collected immediately after birth (n = 4) and after suckling for 6 h (n = 4), 12 h (n = 4) and 24 h (n = 3). Values are means ± SEM, with pluses and asterisks indicating significant differences from values for 0 h pigs for homogenates and BBMV, respectively. Total activity was calculated by summing the products of specific activity times protein content in each region.
[View Larger Version of this Image (20K GIF file)]
Fig. 4.
Total intestinal aminooligopeptidase activity (units, panel A) and specific activity [µmol/(min·mg protein), panel B] for homogenates and brush border membrane vesicles (BBMV) prepared from the proximal (circles), mid (squares) and distal (triangles) small intestines of pigs, collected immediately after birth (n = 4) and after suckling for 6 h (n = 4), 12 h (n = 4) and 24 h (n = 3). Values are means ± SEM, with pluses and asterisks indicating significant differences from values for 0 h pigs for homogenates and BBMV, respectively. Total activity was calculated by summing the products of specific activity times protein content in each region.
[View Larger Version of this Image (20K GIF file)]
Fig. 5.
Aldohexose and amino acid uptake [pmol/(s·mg protein)] by brush border membrane vesicles prepared from the proximal, mid and distal small intestines of pigs, collected immediately after birth and after suckling for 6, 12 and 24 h (n = 2 for each age).
[View Larger Version of this Image (38K GIF file)]
Fig. 6.
Glucose cotransport by intact tissues from the proximal, mid and distal small intestines of pigs, collected immediately after birth (n = 4) and after suckling for 6 h (n = 4), 12 h (n = 4) and 24 h (n = 3). Panel A: Intestinal Na+/D-glucose cotransport capacity (µmol/min). Panel B: Maximum rates of cotransport [nmol/(min·mg tissue) at 50 mmol/L glucose]. The asterisk indicates a difference from the value for 0 h pigs. Transport capacities were calculated by summing the products of maximum rates of uptake in each region times total regional wet weight.
[View Larger Version of this Image (23K GIF file)]
, Widdowson et al. 1976, Xu et al. 1992). However, the gain in intestinal weight as a percentage of body weight during the first 24 h after birth is comparable. The present results show that most of the growth occurs during the first 6 h of suckling and is caused by a rapid increase in the mucosal component of all three regions. This is evident from the increase in gut thickness (Table 1), which results in a 100% increase in absorptive area at the microscopic level (Burrin et al. 1994, Xu et al. 1992) and greatly exceeds the 27% increase in nominal surface area. Although onset of suckling induces similar rapid weight gains in neonates of other species [rats (Berseth et al. 1983), rabbits (Gall and Chung 1982), dogs (Schwarz and Heird 1994)], this influence differs from the low rates of mucosal weight gain in guinea pigs (Weaver and Lucas 1987) and cats during the first week after birth (Buddington 1992).
Protein content.
Mucosal weight gains were accompanied by increases in protein content of the homogenates. This can be attributed to a combination of three factors. First, endocytosis of colostral immunoglobulins, mainly immunoglobulin G (Kiriyama 1992), and nonselective endocytosis of lumenal solutes (Ekstrom and Westrom 1991) would increase homogenate protein content. These activities may be enhanced by protease inhibitors present in colostrum (Lindberg 1982) and the higher protein content of colostrum (13% at birth vs. 6.4% 24 h later; Klobasa et al. 1987). The resulting formation of protein-dense intracellular granules in neonatal enterocytes coincides with changes in cell ultrastructure and a transient epithelial swelling (Xu et al. 1992, Komuves et al. 1993). Although endocytosis and transfer of immunoglobulins to the circulation cease shortly after pigs are born (Westrom et al. 1989), the granules remain for days (Komuves et al. 1993). Second, colostrum increases concentrations of intestinal RNA (Simmen et al. 1990) and protein synthesis by enterocytes that line the intestine at birth (Burrin et al. 1992). Third, higher DNA content at 24 h (Xu et al. 1992, present study) indicates that colostrum stimulates crypt cell proliferation. In preliminary studies using 5-bromo-2
-deoxyuridine, we have confirmed
there is an increase in cell proliferation after 6 h of suckling compared with unsuckled neonates (unpublished data). The different age-related patterns of DNA increase observed for the three regions show there are both spatial and temporal differences in rates of enterocyte proliferation during the first 24 h after birth. The onset of suckling immediately stimulates enterocyte proliferation in the mid and distal small intestine, but the response is delayed in the proximal intestine until after 6 h. An unknown fraction of the increased mucosal DNA would be contributed by adherent bacteria that rapidly colonize the intestine after birth (Swords et al. 1993).
, Puchal and Buddington 1992, Schwarz and Heird 1994), there are shifts in other intestinal functions of pigs. These include declines in the number of receptors for epidermal growth factor (Kelly et al. 1992), loss of receptor-mediated transcytosis of colostral immunoglobulins (Westrom et al. 1989) and an increase in the concentrations of fatty acid binding proteins (Reinhart et al. 1992). The earlier and more dramatic changes in hydrolase activities and rates of transport by proximal intestine correspond with an earlier loss of endocytic capacities relative to distal intestine (24 h vs 6 d; Ekstrom and Westrom 1991) and reflect the proximal to distal pattern of maturation. These changes are not unique to pigs and are reported for other species with rapid postnatal intestinal growth (Buddington 1994, Schwarz and Heird 1994).
, Koldovsky et al. 1992), increases transport of electrolytes and nutrients (Bird et al. 1994), stimulates secretion of glucoregulatory hormones (Burrin 1992, Lepine et al. 1989) and influences later development of the intestine (Kelly et al. 1993). It is commonly thought these influences are mediated by the high concentrations of various growth factors. However, nutrients in colostrum may also be influential, whether directly or indirectly by stimulating secretions.
) and rates of nutrient transport per milligram of tissue, total intestinal lactase activity remains stable (Montgomery et al. 1991) or increases (present study), and transport capacities for most nutrients increase (Buddington 1992). For example, total lactase activity associated with the BBMV of newborn pigs is sufficient to digest 1.7 g of lactose in 1 h, exceeding the 0.34 g of lactose in the 10 g of colostrum (Widdowson 1985) consumed by newborn
pigs (our unpublished observations). Similarly, total lactase activities in the proximal intestine of neonatal puppies (0.25 g/h; Schwarz and Heird 1994) are sufficient to hydrolyze estimated dietary loads (0.15 g; based on consumption of 5 mL milk/h with 3% lactose; Jenness and Sloan 1970). The postnatal increase in total intestinal lactase activity should be more than enough to compensate for increases in milk consumption by pigs during the first 24 h after birth.
), albeit at low activities, suggests pigs are provided with a limited capacity to process alternative sources of carbohydrate. Humans are also born with maltase and sucrase (Auricchio and Sebastio 1989), and this may be an important adaptation that allows neonates of both species to digest foods other than milk and be weaned at or shortly after birth (Greer and Apple 1991, Leibbrandt et al. 1977). Corresponding with the ability of the sucrase-isomaltase and maltase/glucoamylase complexes to hydrolyze maltose, maltase specific activity was higher than that of sucrase. However, diarrhea results when dietary loads exceed total intestinal maltase and sucrase activities, such as we found when feeding neonatal pigs milk replacers high in sucrose. It is difficult to calculate dietary loads of substrates for AOP. However, the presence of AOP in neonatal pigs (present study, Tarvid et al. 1994) and subsequent increase after onset of suckling suggest it is important for hydrolyzing and making available components of milk.
) and for 10 d sucklings (13-fold), and adults (eight-fold, but 13-fold for sucrase), with lactase enrichments at 3 d intermediate (4.9-fold, unpublished data). These findings and the low enrichments for unsuckled neonates (present study, Buddington and Malo 1996) indicate there are changes late in gestation and during the first postnatal days in the insertion of hydrolases into the BBM. The apparent presence of soluble lactase and sucrase, as revealed by assaying the different fractions during BBMV preparation, corresponds with previous reports of higher proportions of soluble BBM hydrolases during early postnatal development (Galand and Forstner 1974, Reisenauer et al. 1992, Seetharam et al. 1977). The lower recovery of sucrase might be related to a greater proportion of soluble sucrase in neonates relative to adults. This might be caused by a weaker attachment of sucrase to the sucrase-isomaltase complex or the entire complex to the BBM, and possible intracellular activation of the enzyme.
). Furthermore, there is a redistribution of transport from along the entire crypt-to-villus axis at birth (Smith 1988), to the tips of villi, as reported for the distribution of Na+/glucose cotransporters in older animals (Freeman et al. 1993, Hwang et al. 1991). The lack of decline in glucose transport by intact distal intestine, despite greater increases in tissue weight and changes in villus architecture, indicates there was either synthesis and insertion of new transporters or increased activity of those existing at birth.
) and the processing of gene products (Burrin et al. 1994). Colostrum induces synthesis and processing of lactase (Burrin et al. 1992) and, in the present study, was associated with an increase in total intestinal mucosal lactase activity. Increases in total intestinal mucosal activities for maltase, sucrase, and AOP provide further evidence that birth and onset of suckling induce synthesis and processing of BBM hydrolases. What is less clear is whether the changes in gene expression reflect reprogramming of existing enterocytes or the production of enterocytes with adult characteristics that eventually replace the fetal enterocytes (Smith 1988).
). These could influence uptake by altering the functions of individual transporters in isolated BBMV, reduce recovery of BBM with transporters from homogenates, or compromise BBMV integrity.
ACKNOWLEDGMENTS
FOOTNOTES
Manuscript received 10 June 1996. Initial reviews completed 30 July 1996. Revision accepted 22 November 1996.
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