Secondary Logo

Journal Logo

Original Article

Intestinal Brush-Border Membrane Enzyme Activities and Transport Functions during Prenatal Development of Pigs

Buddington, Randal K.; Malo, Christiane*

Author Information
Journal of Pediatric Gastroenterology & Nutrition: July 1996 - Volume 23 - Issue 1 - p 51-64
  • Free


Ontogenetic development of the mammalian intestine can be divided into five phases (1). The first three, organogenesis, differentiation, and growth and maturation, occur during gestation and prepare the intestine for birth, when it assumes full responsibility for providing nutrients to the infant. The fourth phase occurs immediately after birth, when the intestine begins to process milk; the fifth is at weaning, when mammals shift from drinking milk to eating the adult diet, at which time the intestine acquires adult structural and functional characteristics. Although the greatest functional demands are placed on the intestine after birth, there is increasing awareness of the intestine's importance in providing nutrients, and possibly growth factors, to the developing fetus by processing swallowed amniotic fluids (2,3).

Comparative studies of age-related changes in intestinal structure and function have uncovered several patterns of development that are related to species differences in natural feeding habits and degree of precocity at birth (4,5). The best known example is sucrase, which is first detected well before birth in humans (6); at or shortly after birth in precocial species, such as pigs and guinea pigs (7,8); not until weaning in altricial rats and rabbits (9); and never in ruminants, such as cows and sheep (10). Relative to the brush-border hydrolases, much less is known about ontogenetic development of nutrient transporters; most of the data are limited to postnatal development, with an emphasis on weaning. Although studies of intestinal transporter development are restricted to relatively few species, and usually only jejunum, patterns of age-related changes are known to differ between species and nutrients (4,11). A recent synthesis of studies has shown a relationship between when active D-glucose transport is first detected and length of gestation (5). Corresponding with this, transport of D-glucose can be detected in humans at 11 weeks (≈28%) of gestation (12) but not until just before birth (≈90% of gestation) in altricial rodents with short pregnancies.

Despite the importance of prenatal intestinal functions, changes in intestinal transport during gestation are not well understood. We are not aware of any studies that characterize from early gestation to birth the progression of changes for transport of glucose or any other solute. The lack of information is related to the limited availability of human tissues, particularly from the last trimester, and a lack of suitable animal models. Therefore, the principal objective of the present study was to characterize Na+-D-glucose cotransport along the length of pig small intestine, beginning at 43% of gestation (7 weeks postconception) until birth. Rates of transport by brush-border membrane vesicles (BBMVs) were measured using a fast sampling, rapid filtration apparatus that allows for accurate determination of initial uptake rates (13), provides precise estimates of transport characteristics (14), and has proved valuable in defining properties of D-glucose and D-aspartic acid transport in human and rabbit small intestine (15,16). The pig was selected for four reasons: the intestines of humans and pigs share similar structure and function and develop early in fetal life; both species are born at relatively advanced states of development, though pigs are slightly less developed; the relatively large size of fetal and newborn pigs allows for studies of intestinal development throughout the life history; and both species have similar nutritional requirements and undergo comparable shifts in dietary habits from milk to omnivory. For these and other reasons, the pig is recognized as a relevant model for studies of pediatric gastroenterology and nutrition (17). A second, related objective was to determine whether the pig is an appropriate model for studies of prenatal development of intestinal nutrient transport.



Fetal and neonatal pigs were obtained from sows in the Mississippi State University swine herd. All were standard, cross-bred farm pigs of similar genetic backgrounds. Fetal stages were obtained from timed-pregnancy sows slaughtered at the Mississippi State University Meat Laboratory. After the sows were killed by electrocution and exsanguination, the uteri were removed intact and cut open, and fetal pigs were removed. Hearts of the fetuses were observed to be beating at the time of death (decapitation) and removal of the intestine. Neonatal pigs were obtained immediately after birth, before suckling, and were killed by Beuthanasia (0.25 ml/kg; i.v.) before removal of the intestine.

Collection of Tissues and Sample Sizes

A total of 28 fetal pigs were collected at weeks 7 (n = 8), 8 (n = 8), 10 (n = 6), and 12 (n = 6) of gestation (from two, two, two, and one sows, respectively). These ages correspond to 43, 49, 61, and 74% of gestation (full term is 114 days). The fragile small intestines of 7-, 8-, and 10-week fetal pigs were frozen intact in liquid nitrogen and stored at -80°C. Because the intestines of fetal pigs are small, it was necessary to pool intestines from two to four fetuses for each BBMV preparation. Specifically, at 7 weeks of gestation, four fetuses originating from each of the two sows were used to prepare two batches of BBMVs, with each batch made using the entire intestines of all four fetuses. At both 8 and 10 weeks of gestation, a sufficient number of fetuses were available from one sow to make two batches of BBMVs, with a single batch of BBMVs prepared from fetuses obtained from the other sow, for a total of three batches of BBMVs from two sows at each age.

Small intestines were collected from six 12-week fetal pigs that originated from a single litter. Each intestine was separated into three regions of equal length (proximal, middle, and distal), frozen in liquid nitrogen, and stored at -80°C. Tissues from two fetuses were pooled to prepare BBMVs. Intestines from five neonatal pigs from two litters were similarly separated into three regions; flushed with cold, aerated mammalian Ringers, after which the mucosa was removed by gently scraping with a glass slide; frozen in liquid nitrogen; and stored at -80°C. Mucosa samples were processed individually, yielding five batches of BBMVs for each region. Therefore, at 7, 8, 10, and 12 weeks of gestation, and at birth two, three, three, three, and five BBMV batches were used to measure enzyme activity and rates of transport by either the entire intestine or the three regions.

Preparation of BBMVs

The frozen tissues were shipped on dry ice to the University of Montreal, where BBMVs were prepared and measurements of glucose transport and hydrolase activities were conducted. BBMVs were prepared by the MgCl2 precipitation method of Hauser et al. (18). The second pellet was suspended in 50 mM Tris-HEPES buffer, pH 7.5, containing 0.1 mM MgSO4, 200 mM KCl, and 125 mM D-mannitol. Aliquots were stored in liquid nitrogen (<2 weeks) until the day of uptake measurements, when the final centrifugation steps of BBMV preparation were done, as previously described (19). BBMVs were suspended in the same medium to a protein concentration of 10-40 mg/ml and 25- or 50-μl aliquots were rapidly frozen in liquid nitrogen until the time of assay (within 48 h of BBMV preparation).

Although preparing BBMVs by this procedure has been shown to prevent loss of transport functions using intestinal tissues from prenatal and postnatal stages of some mammals (15, 20-22), similar validation studies have not been performed for pigs. Therefore, we obtained from a nearby processor a total of 25 fetal pigs at three different stages of gestation; early (10-12 cm), middle (15 cm), and late (17-20 cm). BBMVs were prepared from the entire intestines of the early and mid-gestation fetuses and from the proximal and distal halves of the late gestation pigs (two preparations were made for each age). Initial rates of glucose transport were measured at 25°C immediately after preparation of BBMVs (fresh) and after freezing at -80°C.

Transport Measurements

Transport measurements were done by the rapid filtration technique of Hopfer et al. (23) using a fast sampling rapid filtration apparatus (13) with fully automatic injection of BBMVs (20 μl), mixing of BBMVs with incubation media (480 μl), filtration of ≤18 aliquots of the BBMV-incubation medium mixture, and multiple rinses with stop solution. Compositions of the incubation media are provided in the legends for the tables and figures. Temperature of the incubation mixture was maintained at 15, 25, or 35°C by a recirculating water bath controlled by a thermoprobe inserted into the incubation chamber. At user-defined intervals, aliquots of the BBMV-incubation mixture (50 μl) were injected into 1 ml of ice-cold stop solution containing 50 mM Tris-HEPES buffer (pH 7.5), 0.1 mM MgSO4, 200 mM NaCl, 125 mM D-mannitol, and either 1 mM phlorizin for D-glucose uptake or 50 mM HgCl2 for L-leucine uptake and filtered using 0.65-μm prewetted nitrocellulose filters (Micro Filtration System, Dublin, CA, U.S.A.). After an additional two rinses, each with 1 ml ice-cold stop solution, the filters were dissolved in 5 ml Beta-Blend (ICN Radiochemicals, Irvine, CA, U.S.A.), and radioactivity was determined by liquid scintillation counting (Minoxi Tri-Carb 4000; United Technologies Packard, Downers Grove, IL, U.S.A.).

Data Analysis

Transport data are expressed as picomolar solute uptake per milligram protein. The relationship between incubation time and accumulation of isotope by BBMVs was defined using 18 aliquots taken over a 6-h period. Initial rate determinations were then made using nine aliquots taken over 4.5, 10, or 15 s, depending on rates of isotope accumulation as functions of incubation time. Initial rates of transport were estimated by linear regression analysis over the linear part of the uptake-time curves or by second-degree polynomial analysis when uptake-time curves deviated from linearity (24,25), with the initial rate represented by the first degree coefficient of the polynomial. The kinetic parameters of D-glucose uptake (values ± SD of regression) were determined by nonlinear regression analysis of a displacement curve (15). Different model equations corresponding to either one or two Michaelian saturating components working in the presence or absence of a nonspecific component were tested for each set of kinetic data. Woolf-Augustinsson-Hofstee linear transformations of the data are shown in the figure insets. In all cases, data were analyzed using the Enzfitter program (Biosoft) and an IBM PC-compatible microcomputer with explicit weighting of the data points using the standard deviation of the regression.

Enzyme and Protein Assays

Activities of lactase, sucrase, and hexokinase were measured using the methods of Kunst et al. (26). γ-Glutamyltranspeptidase (γ-GT; EC activity was measured according to Wahlefeld and Bergmeyer (27), using γ-glutamyl para-nitroanilide as the substrate. Protein content of the homogenates and BBMVs was determined using the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL, U.S.A.) and bovine serum albumin as the standard.


All salts and chemicals for buffer preparation were of the highest purity available. D-[1-3H(n)] glucose (15.5 ci/mmol) and L-[4,5-3H(n)]leucine (60 ci/mmol) were purchased from New England Nuclear (Mississauga, Ontario, Canada). Phlorizin was obtained from Aldrich Chemical Co. (Milwaukee, WI, U.S.A.).


Intestinal Weight and Protein Concentrations

Intestinal weight increased exponentially between 7 and 12 weeks of gestation (Fig. 1A), with an even more dramatic increase during the last 4 weeks of gestation (more than sevenfold). During fetal development, protein concentrations of tissue homogenates remained relatively constant, ranging from 70 to 78 mg protein per gram of intact intestine (Fig. 1B). Homogenate protein concentrations of neonatal pigs were higher (115 mg per gram). Protein concentrations of BBMVs (Fig. 1B, filled circles) and recoveries (Fig. 1B, numbers) were higher at 8 weeks relative to 7 and 10 weeks. After 10 weeks there was a dramatic increase in protein recoveries (over two-fold relative to 10 weeks), corresponding with higher BBMV protein concentrations. The higher protein content in homogenates and BBMVs of neonates may be related to the use of mucosa instead of intact tissues. However, protein recovery is comparable to that at 12 weeks.

Enzyme Activities

Sucrase activity was not detected in homogenates or BBMVs from fetal and newborn pigs (Table 1). In contrast, lactase was present in homogenates and BBMVs prepared from 7-week fetal pigs at levels of activity comparable to those measured at 8 and 10 weeks of gestation. BBMV lactase activity increased nearly fourfold between 10 and 12 weeks of gestation and an additional 2.5-fold by birth and established the typical proximal-to-distal gradient of activity. γ-GT was detected in 8-week fetal pigs (not assayed in 7-week fetuses), and activity remained stable throughout gestation, with higher activity in proximal intestine at all ages for which regional data were available. Higher homogenate enzyme activities and lower purification factors for neonatal pigs compared with fetal stages is attributed to the use of mucosal scrapings. The cutosolic enzyme hexokinase was detected in the intestines of all pigs, with a 2.7-fold increase in activity between 12 weeks and birth.

There were two distinct peaks for recovery of lactase and γ-GT when calculated using total activity of BBMVs and homogenate fractions (Fig. 1C). Recoveries for lactase and γ-GT were higher at 8 and 12 weeks of gestation, coinciding with the peaks in BBMV protein recovery (Fig. 1B). Differences between recoveries for lactase and γ-GT at 10 and 12 weeks of gestation are suggestive of enzyme-specific ontogenetic patterns of biosynthesis and insertion into the brush-border membrane.

Validating the Use of Frozen BBMV Preparations

Rates of uptake by frozen BBMV averaged 96 ± 8% of those of fresh, which were consistent for the three fetal stages. These validation results show that rates of transport are accurately determined using frozen BBMVs, thereby allowing for comparisons of different experimental conditions and BBMV preparations. Moreover, the validation studies provide further evidence that rates of uptake increase between early and late gestation, with a strong declining proximal-to-distal gradient of uptake present by late gestation.

Uptake-Time Relationships for D-Glucose

Although uptake-time relationships at most ages exhibited a typical overshoot phenomenon, because of the long time scale (6 h), rapid and tissue-specific rates of accumulation, and temperature effects, all graphical representations use a log scale for incubation time (Figs. 2-4). This approach improves visualization of uptake during the first minutes of incubation, estimates of time of peak overshoot, and appraisal of temperature effects.

D-glucose uptake was measurable in 7-week fetuses. However, accumulation was slow, reaching a maximum of ≈20 pmoles per mg protein after 360 min at 15°C or 30 min at 25 and 35°C, with initial rates of uptake increasing slightly with temperature (Table 2). The typical overshoot of D-glucose uptake was first detected at 8 weeks of gestation (Fig. 2B), corresponding with increases in recovery of BBMV protein (Fig. 1B) and lactase activity (Fig. 1C). Temperature had a profound influence on D-glucose accumulation by BBMVs or 8-week fetuses. The peak of the overshoot occurred sooner at higher temperatures, with maximum overshoot values recorded at 30, 3, and 1 min at 15, 25, and 35°C, respectively. The height of the overshoot was also related to temperature, exceeding equilibrium values by 2.2, 4.5, and fivefold at 15, 25, and 35°C, respectively. Furthermore, initial rates of D-glucose uptake (Table 2) and rates of efflux were directly related to temperature.

The transitory decline in BBMV glucose uptake and reduced temperature response at 10 weeks is unexplained, but a similar decline has been reported for fetal guinea pigs (7). Between 10 and 12 weeks of gestation, initial rates of D-glucose uptake increased dramatically (Table 2). Sometime before 12 weeks, a declining proximal-to-distal gradient of uptake (Fig. 3) was established. D-glucose uptake by 12-week fetuses also regained sensitivity to temperature, with overshoots appearing sooner and being of larger magnitude at higher temperatures in the proximal and mid-intestine. At 35°C the overshoot peaked at 40 s for proximal intestine, with a maximal accumulation 11.6-fold greater than the equilibrium value, while at 25 and 15°C, peak accumulations were observed at 2 and 10 min and exceeded equilibrium values by 10- and 7.4-fold, respectively. A reduced temperature response was observed in the mid-intestine, with maximal uptake of ≈80 pmol per milligram protein at both 25 and 35°C. The height of the overshoot in the distal intestine was lower at 35 than at 25°C, an effect that may be related to the faster efflux rate.

Unsuckled neonatal pigs exhibited very high initial rates of D-glucose uptake (Table 2). In the proximal intestine, peak accumulation at 35°C was 240 pmol per milligram protein at 20 s (19.6-fold over the equilibrium value; Fig. 4). Maximum accumulations by mid-intestine were 12.6, 11.7, and seven-fold greater than equilibrium values at 35, 25, and 15°C, respectively. In the distal intestine initial rates of uptake and rates of efflux were higher at 35°C.

Calculations of temperature coefficients (Q10 values) from data in Table 2 provide further insights about age-related differences in the temperature sensitivity of uptake. Rates of uptake by 8- and 12-week fetuses and unsuckled pigs were acutely sensitive to temperature, particularly between 15 and 25°C (Q10 values averaged 6.1; Table 3), whereas uptake by 7- and 10- week fetuses was less sensitive (Q10 values between 15 and 25°C averaged 2.3). Although differences between ages were less apparent for Q10 values calculated between 25 and 35°C, those for 8- and 12-week fetuses and neonates were still higher (2.7 versus 1.7), and were comparable to those for the increase in glucose transport by human jejunum between 25 and 35°C (2.6 ± 0.2) (15).

Intravesicular Volumes

Intravesicular volumes were estimated by using D-glucose equilibrium spaces at 6 h. In 7- and 10- week fetuses, intravesicular volumes were large (4.64 ± 0.56 μl/mg protein, n = 6) and were not affected by temperature. None of the values had declined to a stable plateau, suggesting that unusually high values may be related to slower efflux. In contrast, estimated intravesicular volumes of BBMVs from 8-week fetuses were both smaller and affected by temperature (3.6, 2.2, and 1.5 μl/mg protein at 15, 25, and 35°C, respectively). BBMVs from proximal, middle, and distal small intestine of 12-week fetuses and newborn pigs were similar in size to those of 8-week fetuses, but were less affected by temperature (2.09 ± 0.26, 1.75 ± 0.46, and 1.43 ± 0.22 μl/mg protein at 15, 25, and 35°C, average of all three ages for the three regions). These data indicate efflux rates, which are affected by temperature, determine the time at which equilibrium will be reached and can thereby influence the estimation of intravesicular volumes.

Initial rates of L-leucine uptake

In contrast to D-glucose, initial rates of L-leucine uptake at 25°C were relatively high in 7-week fetuses and remained stable ≤12 weeks of gestation (Table 2). Between 12 weeks and birth, rates of leucine uptake increased and a distal-to-proximal gradient was established.

Na+-dependency of D-glucose uptake

The respective contributions of Na+-dependent and Na+-independent uptake pathways for D-glucose were evaluated at the different stages of development by measuring initial rates of uptake under four different experimental conditions: in the presence of an Na+ gradient (192 mM out); in the complete absence of Na+, in the presence of Na+ and 1 mM phlorizin, and in the presence of excess unlabeled D-glucose (100 mM). All uptakes were measured at 25°C.

The majority of uptake at 7 and 10 weeks of gestation was Na+-independent (Table 4), corresponding to the lack of overshoot. In contrast, 86% of D-glucose uptake by 8-week fetuses was Na+-dependent and could be inhibited by phlorizin or saturating concentrations of D-glucose. At 12 weeks of gestation and birth, D-glucose uptake was almost strictly Na+-dependent, with negligible Na+-independent uptake in all three intestinal regions. Although different batches of BBMVs were used to study Na+-dependency (Table 4) and initial rates (Table 2) of glucose uptake, and despite differences in the Na+ gradient (96 versus 192 mM), ontogenetic patterns were virtually identical. That is, both studies showed a transient peak in uptake at 8 weeks, with an increase between 10 weeks and birth; however, rates of uptake averaged 3.2 times higher, at 192 mM Na+.

Kinetic Parameters of D-glucose Uptake

Previous studies have shown an effect of temperature on the visualization of multiple D-glucose transport systems in guinea pig jejunum (28). Therefore, all kinetic experiments were performed at 35°C. Initial rates of tracer (4 μM) D-glucose uptake with increasing concentrations of unlabeled substrate (from 10 μM up to 100 mM) were estimated using nine points over 4.5 s of incubation. The best fit for transport data of 7-, 8-, and 10-week fetuses (Table 5) was obtained using an equation that included two transport sites plus diffusion. Other models were rejected because of divergence, negative parameter values, or systematic deviations of the residuals from a random distribution (29).

At 7 weeks of gestation, Vmax values were low for both a high-affinity, low-capacity system and a low-affinity, high-capacity system. The transitory increase in glucose uptake with clear overshoot at 8 weeks was associated with higher Vmax values and improved resolution of both the low- and high-affinity systems. The goodness of fit is evident in Fig. 5A, and the presence of two transport systems is visualized by the curvilinearity of the Woolf-Augustinsson-Hofstee plot (inset). The decline in rates of glucose uptake between 8 and 10 weeks was caused by a reduced Vmax for the low-affinity system, despite a nearly twofold increase in the Vmax for the high-affinity, low-capacity system.

Kinetic data for the proximal and middle intestine of 12-week fetuses could be resolved only by the equation for two sites plus diffusion (Fig. 5B). However, Km values for the low-affinity site were lower than those for younger fetuses (Table 5). Only the high-affinity system was present in distal intestine at 12 weeks of gestation. In proximal and distal intestine of unsuckled pigs only the high-affinity system was clearly defined (Table 5; Fig. 5C, inset), but it was possible to resolve high- and low-affinity systems in the middle intestine with kinetic characteristics similar to those of 7-, 8-, and 10-week fetuses. A small, but significant passive diffusion component was always measured at 35°C, as previously observed in the small intestines of humans (15), guinea pigs (21), and rabbits (24).


Although functional characteristics of the intestine are known to change between early gestation and adulthood, our results provide some of the first insights about the progressive changes in intestinal hydrolase activity and nutrient transport that occur during gestation. In the following sections we describe prenatal intestinal development in the pig based on the use of BBMVs, compare our findings with those for humans, and discuss the relevance of the pig for further studies of prenatal intestinal development.

Age-Related Changes in Intestinal Development of Pigs

Even though the pig is considered an excellent model for studying intestinal development and nutrition (17), most of what is known is limited to postnatal stages (8, 30, 31). The presence of lactase, γ-GT, and hexokinase activity, as well as leucine uptake and Na+-dependent D-glucose transport at 7-8 weeks of gestation in pigs indicates that the first two phases of intestinal development, organogenesis and differentiation, occur before 43% of term.

Prenatal development of the pig intestinal brushborder membrane (BBM) appears to be a discontinuous process, leading up to high rates of uptake at birth. Measurements of BBMV protein, hydrolase activities, and glucose transport are all consistent in showing that there are two critical periods. The first occurs between 7 and 8 weeks of gestation and is characterized by an increase in Na+-D-glucose cotransport, evident from studies of initial rates, kinetics, and Na+ dependency. Whether this period coincides with the onset of fetal swallowing of amniotic fluid and is the reason for the subsequent decline between 8 and 10 weeks in protein content and glucose transport remains unclear. A second, more dramatic period between 10 and 12 weeks of gestation results in higher protein content, lactase activity, and glucose transport. Lactase activity and rates of glucose transport continue to increase between 12 weeks and birth. Although the magnitude of increase is less, by the time of birth, rates of transport by BBMVs are higher than at any other stage of development studied, including after birth (our unpublished data). In a similar manner, rates of uptake for most nutrients by the intestines of pigs and other mammals are highest at birth (4,11,30), which is consistent with the requirements of neonates for large quantities of energy and nutrients on a per kilogram basis.

In contrast to lactase and glucose transport, there were only slight prenatal changes in γ-GT activity and rates of leucine uptake. The dissimilar findings indicate that the various BBM proteins undergo different temporal patterns of synthesis and insertion into the BBM during gestation and are analogous to the well-known different postnatal patterns of gene expression for lactase and sucrase (32).

When the rapid intestinal growth between 12 weeks and birth (nearly sevenfold) is considered in conjunction with hydrolase activities and rates of transport, total intestinal lactase activity and capacities to transport glucose effectively increase by ≈17- and 12-fold, respectively. Intestinal growth also results in increases of total γ-GT activity and leucine transport between 12 weeks and birth, despite the relative lack of change per unit tissue. Furthermore, when hexokinase activity is integrated with intestinal size, there is an increase in activity that corresponds to the capacities to transport glucose. Collectively, the dramatic increases in capacities to hydrolyze and transport nutrients indicate that the last 4 weeks of gestation are critical; during this time the intestine acquires the abilities necessary for processing the first swallows of milk.

The use of BBMVs and nonlinear regression analysis has been instrumental for characterizing Na+-coupled D-glucose transport in the intestines of 17-to 20-week human fetuses (19,22,33) and adult jejunum (15) and late gestation and early postnatal guinea pigs (21). Using the same approach, we detected the presence of both high- and low-affinity systems during early gestation of pigs and a shift to a single high-affinity pathway in proximal intestine at birth. Unfortunately, the use of the entire intestine of 7-, 8-, and 10-week fetuses prevented an assessment of regional characteristics before 12 weeks of gestation. As a result, we do not know whether the low-affinity system is present in the distal intestine before 12 weeks and is subsequently lost or whether it never develops. At least for the proximal intestine, the 12th week of gestation is a transitory phase. Although at 12 weeks, kinetic parameters of D-glucose transport in the proximal intestine were poorly resolved by the equation for two sites, they could not be solved by the one-site equation, whereas by birth only a pure Michaelis-Menten behavior was observed.

These data suggest that the low-affinity carrier is lost from the proximal intestine during the last weeks of gestation, but the actual reasons for the switch in transport systems are unclear. Other possibilities include changes in the intrinsic properties of a given cotransport mechanism or variations in the number and arrangement of subunits of a multimeric carrier (24). Alternatively, the low-affinity system may represent a Na+-independent pathway that is present in the intestinal BBM only at specific stages of development. Further studies of the D-glucose transport system(s) in terms of substrate specificity and Na+/glucose stoichiometries, as reported for human fetal cotransport systems (19,33), will provide a better understanding of the underlying mechanism for the shifts in glucose transport characteristics during gestational development of the pig.

Comparisons with Human Intestinal Development

Humans, the only species for which comparable prenatal data are available, share with pigs several similarities in patterns of intestinal development during gestation. We draw attention to four. First, the intestines of both species develop functional abilities early in gestation. In humans, intestinal disaccharidases and peptidases have been detected as early as 10-11 weeks postconception, corresponding with ≈25% of gestation (6,34), with the declining proximal-to-distal gradient for lactase established before birth (34). Although less is known about prenatal transport functions in human fetuses, the capacities to transport nutrients are known to develop early in gestation (12,19,33,35,36), and by 17-20 weeks of gestation (43-50% of term) proximal-to-distal gradients are established for transport of D-glucose (22) and L-leucine (37). Our findings indicate that the capacities to transport glucose and leucine develop before 43% of gestation in pigs and proximal-to-distal gradients for glucose transport, lactase, and γ-GT are established before 12 weeks (73% of gestation). In contrast, intestinal differentiation and development of brush-border functions do not occur in rodents until nearly 90% of gestation (5).

Second, similarly to the age-related shifts in D-glucose transport characteristics observed in pigs, two Na+-dependent D-glucose transport systems can be resolved in 17- to 20-week human fetuses (19,33), but only a single system in adults (15). Unfortunately, a comparable study of Na+-D-glucose cotransport characteristics from the early stages of gestation to birth has yet to be performed in humans and is unlikely because of limited tissue availability.

Third, the increasing rates of transport during gestation of pigs agree with measurements of initial rates of uptake and transmural potential differences using human intestine (15,22,36), with the agerelated increases in Vmax for the high-affinity carrier suggestive of increasing transporter densities. Similarly, both species exhibit dramatic increases in lactase activity during gestation, particularly during the last few weeks before birth (38), presumably owing to increased gene expression.

Fourth, although intestinal growth of human fetuses is poorly documented, recent observations of intestinal length (39) indicate that the intestines of human fetuses, like those of fetal pigs, grow substantially during the last weeks of gestation. The combination of intestinal growth and increasing rates of intestinal nutrient transport and activities of hydrolases per unit intestine will therefore dramatically increase the capacities of both species to hydrolyze and transport nutrients.

Despite the above-cited similarities, it needs to be noted that pigs differ from humans in two ways. First, pigs do not develop sucrase until the perinatal period, whereas human fetuses possess high sucrase activity early in gestation. Second, passive immunity is acquired transplacentally in humans but after birth in pigs (40). Interestingly, preterm human infants, like newborn pigs, are capable of acquiring immunoglobulins by intestinal mechanisms (1).

Relevance of Pig Model for the Study of Intestinal Development During Gestation

Because of the numerous similarities, fetal pigs may provide a valuable model to address critical gaps in our knowledge about human intestinal development. Of particular interest are the events that occur during the third trimester, for which we know very little because of the limited availability of human fetal tissue. The rapid intestinal growth and increase in functional capacities between 12 weeks and birth in pigs and measurements of hexose absorption by premature infants (41) are in agreement with the concept of intestinal maturation during late gestation in preparation for the onset of extrauterine nutrition. A relevant model, such as the pig, will provide valuable insights into the signals and mechanisms responsible for the dramatic increases in intestinal dimensions, BBM protein concentrations, activities of brush-border and intracellular enzymes, and rates of Na+-dependent and Na+-independent nutrient uptake.

Dietary inputs influence intestinal structure and function during suckling (42,43) through into adulthood (44). The influences of diet composition are now known to extend back to gestation, when fetuses begin to swallow amniotic fluid (2,3). Recent studies with fetal rabbits indicate that the nutrient composition of amniotic fluids plays a critical role in interacting with the genetic determinants that mediate intestinal development during gestation (45). Because of similarities shared with humans, the pig may provide a more relevant model for exploring the signals and mechanisms responsible for changes in intestinal structure and functions during gestation. This information will provide valuable insights into the treatment and care of premature infants.

Acknowledgment: We recognize the efforts of Dr. Anna Puchal-Gardiner and Hélène Lecavalier and the cooperation of Dr. Nancy Cox (Mississippi State University swine research unit) and Bryan Foods (West Point, MS) for providing the timed-pregnancy sows and fetal pigs.

FIG. 1.
FIG. 1.:
Intestinal weight, protein content, and enzyme recovery during gestation of pigs. A: Intestinal weights were measured at the time tissues were homogenized. B: Protein content of homogenates (open symbols) and BBMVs (filled symbols) for 7- to 12-week fetal pigs (circles). Percentages of homogenate protein recovered in BBMVs are presented above each point; values for 12-week fetuses and unsuckled newborn pigs are averages for the three regions of small intestine. C: Percentages of lactase and γ-GT activity recovered in BBMVs from the homogenates. Values for 12-week fetuses and unsuckled newborn pigs are averages for the three regions of small intestine.
FIG. 2.
FIG. 2.:
The influence of temperature on total D-glucose uptake. Incubation time (log) relationships for BBMVs prepared from the entire intestines of 7-week (A), 8-week (B) and 10-week (C) fetal pigs. A single BBMV preparation was used for each age.
FIG. 3.
FIG. 3.:
The influence of temperature on total D-glucose uptake. Incubation time (log) relationships for BBMVs prepared from the three small-intestinal regions of 12-week fetal pigs. A single BBMV preparation was used for each region.
FIG. 4.
FIG. 4.:
The influence of temperature on total D-glucose uptake. Incubation time (log) relationships for BBMVs prepared from the three small-intestinal regions of unsuckled newborn pigs. One BBMV preparation was used for each region.
FIG. 5.
FIG. 5.:
The influence of increasing concentrations of unlabeled D-glucose on initial rates of total uptake of 4 μM tracer D-glucose by BBMVs prepared from 8- (A) and 12-week (B) fetuses and from unsuckled newborn pigs (C). Insets are Woolf-Augustinsson-Hofstee plots. Kinetic constants are presented in Table 5.


1. Ménard D. Growth-promoting factors and the development of the human gut. In: Lebenthal E, ed. Human gastrointestinal development. Raven Press: New York, 1989:123-50.
2. Buchmiller TL, Fonkalsrud EW, Kim CS, et al. Upregulation of nutrient transport in fetal rabbit intestine by transamniotic substrate administration. J Surg Res 1992;52:443-7.
3. Buchmiller TL, Shaw KS, Chopourian L, et al. Effect of transamniotic administration of epidermal growth factor on fetal rabbit small intestinal nutrient transport and disaccharidase development. J Pediatr Surg 1993;28:1239-44.
4. Buddington RK. Intestinal nutrient transport during ontogeny of vertebrates. Am J Physiol 1992;263:R503-9.
5. Buddington RK. Nutrition and ontogenetic development of the intestine. Can J Physiol Pharmacol (in press).
6. Auricchio S, Sebastio G. Development of disaccharidases. In: Lebenthal E, ed. Human gastrointestinal development. New York: Raven Press, 1989: 451-70.
7. Butt JH, Wilson TH. Development of sugar and amino acid transport by intestine and yolk sac of the guinea pig. Am J Physiol 1968;215:1468-77.
8. Manners MJ, Stevens JA. Changes from birth to maturity in the pattern of distribution of lactase and sucrase activity in the mucosa of the small intestine of pigs. Br J Nutr 1972;28:113-127.
9. Henning SJ. Functional development of the gastrointestinal tract. In: Johnson LR, ed. Physiology of the gastrointestinal tract. New York: Raven Press, 1987:286-300.
10. Shirazi-Beechey SP, Kemp RB, Dyer J, Beechey RB. Changes in the functions of the intestinal brush border membrane during the development of the ruminant habit in lambs. Comp Biochem Physiol 1989;94B:801-6.
11. Buddington RK, Diamond JM. Ontogenic development of intestinal nutrient transporters. Annu Rev Physiol 1989;51:601-19.
12. Koldovsky O, Heringova A, Jirsova V, Jirasek JE, Uher J. Transport of glucose against a concentration gradient in everted sacs of jejunum and ileum of human fetuses. Gastroenterology 1965;48:185-7.
13. Berteloot A, Malo C, Breton S, Brunette M. Fast sampling, rapid filtration apparatus: principal characteristics and validation from studies of D-glucose transport in human jejunal brush-border membrane vesicles. J Membr Biol 1991;122:111-25.
14. Berteloot A, Semenza G. Advantages and limitations of vesicles for the characterization and the kinetic analysis of transport systems. In: Fleischer S, Fleischer B, eds. II. Cellular and subcellular transport: epithelial cells. Orlando, FL: Academic Press, 1990:409-37. (Methods in enzymology: biomembranes).
15. Malo C, Berteloot A. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na+-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J Membr Biol 1991;122:127-41.
16. Maenz DD, Chenu C, Breton S, Berteloot A. pH-dependent heterogeneity of acidic amino acid transport in rabbit jejunal brush-border membrane vesicles. J Biol Chem 1992;267:1510-6.
17. Moughan PJ, Birtles MJ, Cranwell PD, Smith WC, Pedraza M. The piglet as a model animal for studying aspects of digestion and absorption in milk-fed human infants. In: Simopoulos AP, ed. Nutritional triggers for health and in disease. Basel: Karger, 1992:40-113. (World review of nutrition and diet, Vol. 67).
18. Hauser H, Howell K, Dawson RMC, Bowyer DE. Rabbit small intestinal brush border membrane preparation and lipid composition. Biochim Biophys Acta 1980;602:567-77.
19. Malo C. Kinetic evidence for heterogeneity in Na+-D-glucose cotransport systems in the normal human fetal small intestine. Biochim Biophys Acta 1988;938:181-8.
20. Maenz, DD, Chenu C, Bellemare F, Berteloot A. Improved stability of rabbit and rat intestinal brush-border membrane vesicles using phospholipase inhibitors. Biochim Biophys Acta 1991;1069:250-8.
21. Malo C. Ontogeny of Na+/D-glucose cotransport in guineapig jejunal vesicles: only one system is involved at both 20°C and 35°C. Biochim Biophys Acta 1993;1153:299-307.
22. Malo C, Berteloot A. Proximo-distal gradient of Na+-dependent D-glucose transport activity in the brush border membrane vesicles from the human fetal small intestine. FEBS Lett 1987;220:201-5.
23. Hopfer UK, Nelson K, Perrotto J, Isselbacher KJ. Glucose transport in isolated brush-border membrane from the rat small intestine. J Biol Chem 1973;248:25-32.
24. Chenu C, Berteloot A. Allosterism and Na+-D-glucose cotransport kinetics in rabbit jejunal vesicles: compatibility with mixed positive and negative cooperatively in a homodimeric or tetrameric structure and experimental evidence for only one transport protein involved. J Membr Biol 1993;132:95-113.
25. Dorando FC, Crane RK. Studies of the kinetics of Na+ gradient-coupled glucose transport as found in brush-border membrane vesicles from rabbit jejunum. Biochim Biophys Acta 1984;772:273-87.
26. Kunst A, Draeger B, Ziegenhorn J. UV-methods with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Metabolites II. Carbohydrates, 3rd ed. Weinheim: Verlag Chemie, 1984:163-72. (Methods of enzymatic analysis, Vol. 6).
27. Wahlefeld AW, Bergmeyer HU. γ-Glutamyltransferase, routine method. In: Bergmeyer HU, ed. Enzymes II. Oxidoreductases, Transferases, 3rd ed. Weinheim: Verlag Chemie, 1984:352-6. (Methods of enzymatic analysis, Vol. 3).
28. Brot-Laroche E, Serrano MA, Delhomme B, Alvarado F. Temperature sensitivity and substrate specificity of two distinct Na+-activated D-glucose transport systems in guineapig jejunal brush-border membrane vesicles. J Biol Chem 1986;261:6168-76.
29. Mannervik B. Regression analysis, experimental error, and statistical criteria in the design and analysis of experiments for discrimination between rival kinetic models. Meth Enzymol 1982;87:371-90.
30. Puchal AA, Buddington RK. Postnatal development of monosaccharide transport in pig intestine. Am J Physiol (Gastrointest Liver Physiol) 1992;262:G895-902.
31. Smith MW. Postnatal development of transport function in the pig intestine. Comp Biochem Physiol 1989;94B:801-6.
32. Freund JN, Torp N, Duluc I, Foltzer-Jourdainne C, Danielsen M, Raul F. Comparative expression of the mRNA for three intestinal hydrolases during postnatal development in the rat. Cell Molec Biol 1990;36:729-36.
33. Malo C. Separation of two distinct Na+D-glucose cotransport systems in the human fetal jejunum by means of their differential specificity for 3-0-methylglucose. Biochim Biophys Acta 1990;1022:8-16.
34. Lindberg T. Intestinal dipeptidases: characterization, development and distribution of intestinal dipeptidases of the human foetus. Clin Sci 1966;30:505-15.
35. Jirsova V, Koldovsky O, Heringova A, Hoskova J, Jirasek J, Uher J. The development of the functions of the small intestine of the human fetus. Biol Neonat 1966;9:44-9.
36. Levin RJ, Koldovsky O, Hoskova J, Jirsova V, Uher J. Electrical activity across human fetal small intestine associated with absorption processes. Gut 1968;9:206-13.
37. Malo C. Multiple pathways for amino acid transport in brush border membrane vesicles isolated from the human fetal small intestine. Gastroenterology 1991;100:1644-52.
38. Antonowicz I, Lebenthal E. Developmental pattern of small intestinal enterokinase and disaccharidase activities in the human fetus. Gastroenterology 1977;72:1299-303.
39. Shanklin DR, Cooke RJ. Effects of intrauterine growth on intestinal length in the human fetus. Biol Neonate 1993;64:76-81.
40. Westrom BR, Karlsson BW, Ekstrom G, Svendsen J, Svendsen LS. The neonatal pig as a model for studying intestinal macromolecular transmission. In: Tumbleson ME, ed. Swine in biomedial research, Vol. 2. New York: Plenum Press, 1986:1297-301.
41. Murray RD, Boutton TW, Klein PD, Gilbert M, Paule CL, Maclean WC Jr. Comparative absorption of [13C]glucose and [13C]lactose by premature infants. Am J Clin Nutr 1990;51:59-66.
42. Berseth CL, Nordyke CK, Valdes MG, Furlow BL, Go VLW. Responses of gastrointestinal peptides and motor activity to milk and water feedings in preterm and term infants. Pediatr Res 1992;31:587-90.
43. Sheard NF, Walker WA. The role of breast milk in the development of the gastrointestinal tract. Nutr Rev 1988;46:1-8.
44. Ferraris RP, Diamond JM. Specific regulation of intestinal nutrient transporters by their dietary substrates. Annu Rev Physiol 1989;51:125-42.
45. Trahair JF. Is fetal enteral nutrition important for normal gastrointestinal growth? A discussion. J Parenteral Enteral Nutr 1993;17:82-5.

Fetal; Uptake; L-leucine; Na+-D-glucose cotransport; Kinetics; Lactase; Ontogeny; Gestation

© Lippincott-Raven Publishers