rates of leucine and proline uptake, despite identical types and concentration of protein in the two milk. M*1 l'O ss. d o cs PNPO. >c POPO. >es csooo pooo ooo.
Nutrient Metabolism
Intestinal Amino Acid and Monosaccharide Transport in Suckling Pigs Fed Milk Replacers with Different Sources of Carbohydrate1 YVONNE
M. VEGA, ANNA
A. PÃœCHAL AND RANDAL
K. BUDDINGTON2
Department of Biological Sciences and College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762-5759 bilities can also change during a species' life history. For example, as omnivorous tadpoles, frogs exhibit dietary modulation of glucose uptake (Toloza and Diamond 1990) but lose this adaptive ability during or after metamorphosis to the adult stage (Ferraris and Diamond 1989). Mammals undergo age-related shifts in diet that are quite different from those in frogs. As sucklings, mammals consume a diet (milk) that is relatively constant in composition over time. Only after weaning is the diet of mammals likely to vary. Be cause the composition of milk has probably remained constant during a species' phylogeny, suckling mammals, like carnivores, may lack or have a reduced ability to adaptively modulate rates of uptake in response to changes in dietary composition. Rates of intestinal nutrient uptake change during suckling development, despite dietary constancy (Buddington 1992, Buddington and Diamond 1989). What remains unclear is whether dietary inputs can influence the age-related changes. This lack of infor mation is surprising when one considers that many, if not most, infants worldwide are weaned at birth or shortly thereafter to either solid foods or infant for mulas that can differ markedly from breast milk in levels and sources of nutrients (Greer and Apple 1991). To address this lack of information, we measured rates of sugar and amino acid uptake by the intestines of suckling pigs fed milk replacers with either lactose or a 60:40 mixture of maltodextrin and sucrose as the source of carbohydrate,- the milk replacers were otherwise identical. We chose the pig as the model because of five important attributes. First, because pigs are large at birth (~1 kg) with large intestines
INDEXING KEY WORDS:
•pigs •intestine •transport •diet •development
The abilities of vertebrates to adaptively modulate rates of nutrient transport are related to each species' natural feeding habits. Whereas omnivores can adap tively modulate rates of transport to match changes in quantities and types of carbohydrate (Ferraris and Diamond 1989), representatives of exclusively car nivorous families have either lost or never acquired such abilities (Buddington et al. 1991). Adaptive capa 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
'Supported in part by Milk Specialties, the Carnation Company, a Mississippi State University Research Initiation Award to R. K. Buddington and a CIRIT (Catalunyan) fellowship to A. A. Puchal. 2To whom correspondence should be addressed.
Received 11 May 1992. Accepted 25 August 1992. 2430
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ABSTRACT Omnivorous mammals are able to adaptively modulate rates of intestinal nutrient transport to match changes in diet. Because adaptive responses during suckling, when dietary composition is relatively constant, have not been adequately determined, we measured in vitro sugar and amino acid uptake [nmol (mg tissue-min)) in suckling pigs fed milk replacers with either lactose (LAC) or a 60:40 mixture of maltodextrin and sucrose (MDS). The MDS-fed pigs initially grew slower, but had intestinal dimensions similar to those of LAC-fed siblings when normalized to body weight. Carrier-mediated uptake for three monosaccharides (glucose, galactose, fructose) did not differ between LAC- and MDS-fed pigs at 5, 10, 15 and 20 d of age. Interdict differences in rates of leucine and proline uptake, despite identical types and concentration of protein in both milk replacers, are indicative of non specific responses to diet during suckling, uptake ca pacities (grams of monosaccharide absorbed per 24 h) never exceeded estimates of monosaccharide intake by more than fourfold and were less than aldohexose intake during early suckling. Our results indicate /) age-related changes in rates of nutrient uptake are genetically pro grammed and little influenced by diet; 2) any responses to diet are nonspecific and likely involve a shift in the timing of the genetic program; and 3) at birth and throughout suckling, pigs are capable of absorbing limited quantities of alternative nutrients. J. Nutr. 122: 2430-2439, 1992.
DIET AND NUTRIENT
UPTAKE IN SUCKLING
(about 3 m long and 0.5 cm in diameter at birth), we were able to measure nutrient uptake at multiple sites along the small intestine. Second, pigs have large litter sizes (8-12 per litter), which allowed us to compare rates of transport by siblings fed the two milk replacers at several ages. Third, baby pigs readily accept milk replacers, which allowed us to avoid the artificial rearing methods required for handling suckling rodents; these methods are difficult and cause stress-related intestinal maturation (Goda et al. 1985, Patel and Hiremagalur 1992). Fourth, pigs and humans share similar intestinal structure, function, shifts in dietary habits and patterns of development, although pigs are born at a slightly earlier stage than humans. Finally, because both pigs and humans differ from more commonly used animal models (e.g., ro dents) in having flexible weaning ages, our results should be relevant to human infants fed foods other than breast milk.
PIGS
2431
TABLE 1 Composition
of the experimental
Component
milk replacers1 LAC diet
MDS diet
g/kg dry mixture 194 113 143 550 0
Experimental base Sodium cascinale Lactose-free whey Lactose 60:40 Maltodextrin:sucrose
194 113 143 0 550
'The components were mixed and stored dry, and were hydrated
MATERIALS AND METHODS Animals. All experimental procedures were ap proved by the Mississippi State University Institu tional Animal Care and Use Committee. We used a total of 55 crossbred farm pigs of both genders from 12 litters obtained from the swine herd maintained by the Mississippi State University Swine Research Unit. From each litter we selected four to eight pigs of similar body weights. The pigs were allowed to suckle for 36-48 h for acquisition of passive immunity via colostrum before they were removed from sows and transferred to the College of Veterinary Medicine. Each litter was divided into two equal groups and placed in 0.9 x 1.2 m cages (Agriplastics, Goshen, IN) provided with overhanging heating lamps in a room maintained at 27-29°C. Pigs in each cage were fed one of two milk replacers that contained either lactose as the sole source of carbohydrate (LAC diet)3 or a 60:40 mixture of maltodextrin (Maltrin 100, Grain Processing Corp., Muscatine, IA) and sucrose using cane sugar (MDS diet). All other components of the diet were identical (Table 1). We used lactose-free whey to ensure that the MDS diet was devoid of lactose and that both diets contained identical amounts of carbohydrate. The milk replacers were made fresh three times each day and fed using self-filling feeders. Sampling. Each pig was weighed daily and general condition recorded. Rates of sugar and amino acid uptake were measured in five pigs from separate litters at the time they were removed from the sows (2 d old) for initial values. We sampled pigs from each dietary group at 5, 10, 15 and 20 d of age (cor responding with 3, 8, 13 and 18 d of feeding the two milk replacers), using at least five pigs per diet at each age. Final body weights were recorded before the pigs were killed by electrocution and exsanguination. The
small intestine was rapidly resected from the pyloric sphincter to the ileocolonic junction and placed in cold (2-4°C) aerated (95% O2 and 5% CO2) mam malian Ringer's solution. As described previously (Puchal and Buddington 1992), the associated mesen teries were cut so the intestine could be straightened along a table top and the length measured. We re moved segments from three regions of the small in testine: proximal (originating -20 cm distal to the pyloric sphincter), mid (located equidistant from the two ends of small intestine) and distal (-20 cm proximal to the ileocolonic junction). We used 10-cm segments from each region to determine regional weight, surface area and mucosal mass per centimeter (Puchal and Buddington 1992). Measurement of uptake rates. Rates of uptake were measured using a modification of the in vitro everted sleeve method (Puchal and Buddington 1992). Briefly, 10- to 15-cm segments from the three regions were flushed with cold, aerated Ringer's solution and slit along the mesenteric border to open them as a sheet. Tissue patches (~1 cm2) were cut from each segment and secured onto the tips of 0.5-cm stainless steel rods by silk ligatures with the mucosa oriented outward, thereby exposing 0.51 cm2 of mucosa. During and after the mounting procedure, the tissues were kept in cold, aerated Ringer's solution. Measurements of nutrient uptake were started 45 min after death. Tissues were preincubated for 5 min
''Abbreviations
used: LAC, lactose-based
maltodextrin-sucrose-based glycol.
milk
replacer;
milk replacer; MDS, PEG,
polyethylene
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with l L of warm water per 170 g of dry milk replacer before feeding. The experimental base was formulated by Milk Specialties (Dundee, IL| such that the complete experimental milk replacers contained 26% protein, 15% fat and 55% carbohydrate on a dry weight basis. Vitamin levels were (mg/kg dry mix): retinyl palmitate, 6.9; cholecalciferol, 0.14; a-tocopherol acetate, 9.5. Mineral levels were (mg/kg dry mix): calcium, 10,600; phosphorus, 8,500; magnesium, 1200; iron, 105; cobalt, 1.2; copper, 14.2; man ganese, 41.3; zinc, 128.5; selenium, 0.39; iodine, 10.9.
2432
VEGA ET AL.
in 38°Caerated mammalian Ringer's solution before they were incubated for 2 min in aerated, 38°C; Ringer's solution with 50 mmol/L concentrations of
solution (sodium replaced entirely by choline) from rates of uptake by adjacent tissues exposed to amino acid in normal Ringer's solution. Tissues exposed to monosaccharide solutions were rinsed for 20 s in cold (2-4°C),nutrient-free Ringer's solution to reduce radioactivity associated with ad herent fluids (Karasov and Diamond 1983). We did not rinse tissues exposed to amino acid solutions because diffusive characteristics of PEG differ from those of the amino acids. After careful blotting to reduce ad herent fluids, the tissues were cut from the rods, placed in tared vials to determine wet weights and solubilized (TS-2; Research Products International, Mt. Prospect, IL), and scintillant was added (4A20; Research Products International). Radioactivity as sociated with the tissues was determined by liquid scintillation counting and used to calculate uptake rates (Karasov and Diamond 1983). Rates of uptake (nmol/min) for each amino acid and monosaccharide were normalized to wet weight (mg) and surface area (cm2). We present only rates of uptake per milligram, because rates of uptake normalized to surface area yielded the same conclusions. All chemicals used were of the highest purity available (Sigma Chemical, St. Louis, MO). Radioiso topes were obtained from Du Pont NEN (Boston, MA) ([3H]L-glucose, [14C]D-fructose and D-galactose), Re search Products International ([14CJD-glucose, [3H]Lleucine and L-proline) and American Radiolabeled Chemicals (St. Louis, MO) |[14C]PEG).
RESULTS Growth and intestinal dimensions. Whereas LACfed pigs had solid stools throughout the study, MDSfed pigs suffered from mild diarrhea for the first 10 to 12 d of feeding, after which stools became firm. The MDS-fed pigs were smaller (Table 2; P < 0.001 for all ages combined) because of lower growth (g gained/kg body wt) during the first 2 d of feeding (d 2 to 4). Despite diarrhea of MDS-fed pigs, by d 4 and there after, rates of body weight gain did not differ between treatments (data not shown). Corresponding with smaller body sizes, MDS-fed pigs had smaller intestines. However, when intestinal dimensions were normalized to body weights, the two diet groups did not differ from each other (P > 0.50) or from pigs suckled by sows (P > 0.50; Puchal and Buddington 1992). The lack of differences in weightcorrected intestinal dimensions is of critical impor tance because it indicates that MDS-fed pigs were not malnourished (Butzner and Gall 1990) but were simply smaller than LAC-fed pigs. Nutrient uptake. Sugars. Rates of monosaccharide uptake did not differ between treatments at any age, when averaged over the entire small intestine, (Fig. 1), or for comparisons within each region; fructose in proximal intestine at 15 d was the only exception (Table 3). Both groups exhibited parallel declines in monosaccharide uptake between d 2 and 20, but the decline was significant for only fructose (P < 0.05). When all age groups were combined, pigs from both diet groups exhibited proximal to distal declines in rates of uptake for the three monosaccharides. However, the proximal to distal gradient was dis rupted at d 10 for MDS-fed pigs and d 15 for LAC-fed pigs, when rates of uptake in the mid region were similar to, or slightly higher than, those in the proximal region. Proximal to distal gradients were reestablished in both groups by the next sampling date. To identify possible dietary influences that might not be apparent from comparisons of absolute rates of sugar uptake, we also compared ratios for the uptake of galactose and fructose relative to glucose
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sugars and amino acids (by isosmotic replacement of NaCl). The incubation solutions were stirred at 1200 rpm to reduce unstirred layer influences. These condi tions yield rates of uptake by suckling pig intestine within the linear phase, and at or near maximum for monosaccharides (Puchal and Buddington 1992) and amino acids (unpublished data). To quantify uptake of glucose, galactose and fructose, we added trace levels of [14C]D-isomer of each monosaccharide to the respective sugar solutions. Because we used [3H]Lglucose to correct for monosaccharide associated with the extracellular space and passively absorbed, our rates of sugar uptake represent amounts of monosac charide absorbed via carrier-mediated pathways. Rates of leucine and proline absorption were measured by adding trace levels of [3H]L-isomer of each amino acid to the respective amino acid solutions. We used [14C]polyethylene glycol (PEG, molecular weight 4000) to correct for amino acid in the adherent fluids. Because PEG does not freely diffuse through the apical membrane, our rates of leucine and proline absorption represent the sum of both carrier-mediated and passive components of absorption. We estimated the sodium-dependent component of amino acid ab sorption by subtracting rates of leucine and proline uptake by tissues incubated in sodium-free Ringer's
Statistics. Values are presented as means ±SEM. Rates of uptake by pigs fed both diets at different ages were examined by one-, two- or three-way ANOVA to examine for the influences of age, diet and region on rates of uptake, and for possible interactions. Un balanced sample sizes were accounted for using SAS general linear model procedures (SAS Institute, Gary, NC). Age and regional influences were assessed from d 2 to 20, and diet influences from d 5 to 20, when the two groups were fed different diets. P < 0.05 was accepted as the critical level for significance in all tests.
DIET AND NUTRIENT
UPTAKE IN SUCKLING
(galactose:glucose and fructose:glucose ratios). De spite the differences in dietary carbohydrate compo sition, both groups exhibited similar increases in both galactose:glucose and fructose:glucose ratios between d 2 and 5, followed by significant declines between d 5 and 20 (P < 0.03 and P < 0.005 for age-related declines in galactose:glucose and fructose:glucose ratios, respectively). There were no interdict differ ences for the timing and magnitude of changes. Amino acids. The following paragraphs describe the influences of age, diet and region on rates of total, Na+-dependent and Na+-independent uptake of leucine and proline by LAC-fed and MDS-fed pigs. Rates of total leucine uptake (carrier mediated plus passive influx) averaged over the entire length of in testine did not differ between groups at any age (P > 0.50 for interdict comparison of d 5-20) and by d 20 were lower than those at d 2 (Fig. 2A; P < 0.002 for comparison with d 2). When compared with uptakes at d 2, lower total leucine uptakes in both groups at d 20 were caused by age-related declines in rates of Na+dependent (active, carrier-mediated; P < 0.10) and Na+-independent (includes passive influx,- P < 0.05)
independent pathways also differed among the three regions of small intestine, representing >95% of total uptake in proximal and distal intestine, but 87% in mid intestine (P < 0.05 for comparisons of mid with proximal and distal intestine). Total, Na+-dependent and Na+-independent proline uptakes did not vary with age in MDS-fed pigs (Fig. IB) but were all lower in LAC-fed pigs at d 10 and 15 relative to d 5 (P < 0.05 for d 10 and 15 pooled). The LAC-fed pigs had a transient increase in Na+-dependent proline uptake at d 5 (P < 0.05 for comparison with LAC-fed pigs at other ages); Na+-independent uptake did not differ between groups at any age. By d 20, rates of proline uptake were similar in both groups. When all ages and both diets were pooled, regional differences were detected for total and Na+independent proline uptake (P < 0.01; rates of uptake were lower in distal intestine compared with proximal and mid intestine); Na+-dependent uptake did not vary among regions (P > 0.30). To summarize, we detected interdict differences for rates of leucine and proline uptake, despite identical types and concentration of protein in the two milk
2433
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FIGURE 2 Rates of total and Na+-dependent uptake for leucine (A) and proline (B) averaged over the entire intestine of suckling pigs at 2 d and after feeding on experimental milk replacers. The first of each pair of bars represents pigs fed the lactose-based milk replacer (LAC), and the second is for pigs fed the 60:40 mixture of maltodextrin and sucrose (MDS). Values are means + SEM; sample sizes are in Table 2. An asterisk indicates difference between diets at P < 0.05; for total or Na+-dependent.
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DIET AND NUTRIENT
2435
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