Vol. 279, Issue 6, R2287-R2296, December 2000
Intestinal transport of monosaccharides and amino acids during
postnatal development of mink
Randal K.
Buddington1,
Christiane
Malo2,
Per T.
Sangild3, and
Jan
Elnif3
1 Department of Biological Sciences, Mississippi State
University, Mississippi State, Mississippi 39762; 2 Membrane
Transport Research Group, Department of Physiology, University of
Montreal, Montreal, Quebec, H3C 3J7 Canada; and 3 Fur Animal
Science, Department of Animal Science and Animal Health, DK-1870
Frederiksberg C, Denmark
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ABSTRACT |
Intestinal development is
typically studied using omnivores. For comparative purposes, we
examined an altricial carnivore, the mink (Mustela vison).
In mink, intestinal dimensions increase up to 8 wk after birth and then
remain constant (length) or decrease (mass) into maturity despite
continuing gains in body mass. Rates of glucose and fructose
transport decline after birth for intact tissues but increase for
brush-border membrane vesicles (BBMV). Rates of absorption for five
amino acids that are substrates for the acidic (aspartate), basic
(lysine), neutral (leucine and methionine), and imino acid (proline)
carriers increase between birth and 24 h for intact tissues before
declining, but increase after 2 wk for BBMV. The proportion of BBMV
amino acid uptake that is Na+-dependent increases during
development but for aspartate is nearly 100% at all ages. Tracer
uptake by BBMV can be inhibited by 100 mmol/l of unlabeled amino acid,
except for lysine. BBMV uptake of the dipeptide glycyl-sarcosine does
not differ between ages, is not Na+ dependent, and is only
partially inhibited by 100 mmol/l unlabeled dipeptide. Despite the
ability to rapidly and efficiently digest high dietary loads of
protein, rates of amino acid and peptide absorption are not markedly
higher than those of other mammals.
nutrient absorption; ontogeny; carnivore; sugar
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INTRODUCTION |
INTESTINAL STRUCTURE AND
FUNCTIONS change during ontogenetic development of mammals,
either in anticipation of, or in response to, shifts in diet
composition. The changes in intestinal structure and functions that
occur at weaning are the best-known examples. These include a decrease
in villus height, increase in crypt depth, increase in gastric
proteases, and the well-known reciprocal shift in the intestinal
activities of lactase and sucrase (19). The magnitude and
timing of the age-related changes are the result of dietary inputs
interacting with genetic determinants. Corresponding with this,
age-related changes do not occur at the same time for all species or
for the various digestive functions. For example, sucrase and lactase
are expressed early in gestation of human fetuses, and although lactase
is detected before birth in species such as the rat and mouse, sucrase
does not develop until the time of weaning (19).
Patterns of development for the intestinal apical transporters also
vary among species and solutes (3, 12). For example, apical transporters for glucose and amino acids appear during gestation
at the time the enterocytes differentiate, whereas carrier-mediated fructose transport does not develop until the time of birth in most
precocial species, such as pigs, and not until weaning in altricial
rodents, such as rats and mice. Another dramatic example is the
appearance of the bile acid transporters at weaning, with expression
restricted to the distal ileum.
Development of digestive functions are much better known for omnivores
compared with carnivores, which undergo a markedly different shift in
dietary inputs at weaning. The few carnivorous mammals that have been
studied (dog and cat) are born at a relatively advanced stage of
development. Mink provide an interesting comparison for three reasons.
First, mink are born altricial, much like laboratory rats and mice are
born at an early stage of development. However, in the mink, the
digestive tract develops slower, and the sensitivity of hydrolytic
activity to stimulation by glucocorticoids develops later (11,
20). Second, even though juvenile and adult mink are strict
carnivores and in the wild they consume aquatic animals (fish,
crustaceans, amphibians), birds, and mammals up to the size of rabbits,
their intestine is able to transport fructose and can modulate rates of
transport to match changes in dietary levels of protein and
carbohydrate (4). This later ability sets them apart from
cats and may be related to how some members of the Mustelidae
(e.g., skunks) are omnivores and that mink may have retained some
ancestral traits. Third, the intestinal length of the mink is short [4
times body length (10)], like those of other carnivorous
mammals (22), and even though food transits the intestine
rapidly [3-4 h in adults (2, 14)], protein digestibility is high (21).
From these interesting characteristics of the mink, we set out to
characterize age-related changes in the rates and regional distribution
of nutrient absorption. The expectation was that because of the
carnivorous diet, rates of absorption for amino acids and peptides
would be higher for mink compared with omnivores, and also relative to
other carnivores because of the rapid and efficient digestion of the
high dietary loads of protein. This was examined by measuring from
birth to maturity rates of absorption along the entire length of small
intestine for five amino acids that represent substrates for different
carriers that handle acidic (aspartate), basic, (lysine), neutral
(leucine and methionine), and imino (proline) amino acids
(13), for the dipeptide glycyl-sarcosine, and for two
sugars, glucose and fructose, that are carried by two different apical
transporters [SGLT-1 and GLUT-5 (1, 24)]. Absorptive
characteristics of intact tissues were studied using everted sleeves,
whereas sodium dependency of the carriers was examined using
brush-border membrane vesicles (BBMV).
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MATERIALS AND METHODS |
Animals and Their Care
Mink of the mahogany strain were obtained from the Department of
Animal Science and Animal Health of the Danish Royal Veterinary and
Agricultural University (Copenhagen). The use of the animals followed
the guidelines approved by the Member States of the Council of Europe.
The mink were housed in conventional external facilities and exposed to
ambient temperatures and light conditions. A standard production diet
formulated with animal by-products and with 56%, 35%, and 9% of the
calories from protein, fat, and carbohydrate, respectively (unpublished
findings from proximate analyses), was provided to the mink twice each
day. To reduce variation for onset of solid food ingestion, small
amounts of the adult diet were placed in the nest boxes when the kits
were 4 wk old. The kits were allowed to suckle until 6 wk of age at
which time they were separated from the females and thereafter received
only the production diet.
Sampling
Mink kits of both sexes were obtained immediately after birth
before suckling (0 h; n = 17), after suckling for 1 day
(n = 17), at 1 (n = 12), 2 (n = 12), 4 (n = 12), and 6 (n = 12) wk of suckling, and at 8 wk after weaning
(n = 8). Adult females were studied at least 6 wk after
lactation had finished (n = 6). At each age, all of the
kits originated from different litters. The 0-h, 1-day, and 1-wk-old
mink were killed by decapitation. Older mink were sedated with
ketamine hydrochloride (Ketominol Vet; Veteraria, Zurich; 50 mg/kg
im) and xylazine (Rompun; Bayer, Leverkusen; 10 mg/kg im) before
decapitation was performed (11).
After death, the entire gastrointestinal tract was removed and placed
within 1 min in cold (2-4°C) Ringer solution that had been
aerated with a mixture of O2 and CO2
(95%:5%). The intestine was freed from the associated mesentery, and
the length (from the pyloric sphincter to the ileocolonic junction) was
measured on a table top in a relaxed state. Between 24 h and 4 wk
only milk was present in the stomach and intestine, whereas both milk and the solid food were present at 6 wk.
For the 0-h, 1-day, and 1-wk-old mink, two segments of small intestine
were used for measurements of sugar and amino acid transport by intact
tissues. A proximal segment of 12-15 cm was taken beginning from
the end of the attached pancreas. A second segment of 10-12 cm was
taken at about 67% of small intestinal length. The distal-most region
of the neonate intestine was too friable and small in diameter for
everting and measuring uptake by intact tissues. For all other ages the
intestine was separated into three regions of equal length, which were
designated as proximal, mid, and distal, and these were used to measure
rates of uptake by intact tissues. For BBMV studies (2 wk and older),
the small intestine was cut into proximal and distal halves to ensure
availability of adequate amounts of tissue. The two regions were frozen
intact in liquid nitrogen and stored at 
70°C until shipped on dry
ice to the University of Montreal, where they were again held at
70°C until used.
Intact tissue measurements.
Following a previous study (4), we measured nutrient
uptake between 45 and 100 min after the animal was killed by incubating 1-cm everted sleeves for 2 min in mammalian Ringer with 50 mmol/l for
each of the sugars and amino acids. At each age, rates of absorption
for all nutrients were measured using a minimum of six animals that
originated from different litters. Accumulation of glucose and fructose
by the tissues was quantified by adding trace levels of
14C-labeled D-isomers of the sugars to the
incubation solutions. L-[3H]glucose was also
added for simultaneous correction of sugar adherent to the tissue and
absorbed independent of carriers. For measuring amino acid absorption,
the 3H-labeled L-isomer was used and
[14C]polyethylene glycol (4,000 mol wt) was added for
correction of amino acid associated with the adherent fluid. The uptake
solutions were aerated with the gas mixture to maintain tissue
viability and stirred (1,200 rpm) to minimize unstirred layer effects.
After the incubation, tissues exposed to the sugars were rinsed for 20 s in cold Ringer, but not those incubated in the amino acid solutions. After tissue mass was recorded, solubilizer (Optisol, Wallach Biochem) and scintillant (Optisafe 2, Wallach Biochem) were
added, and radioactivity was measured by liquid scintillation counting.
Calculated rates of transport (15) were normalized to
tissue mass. Presented values for glucose and fructose represent carrier-mediated uptake, whereas those for the amino acids are the sum
of the carrier-mediated and carrier independent components of absorption.
The regional distribution of absorption was examined by incubating
sleeves from each of the different regions in 50 mmol/l solutions of
the nutrients. The relationships between glucose and fructose
concentrations and rates of uptake were defined by incubating tissues
from the proximal small intestine (region of highest sugar uptake) (4)
in solutions containing tracer alone and in the presence of 0.1, 1, 10, and 50 mmol/l of unlabeled sugar for mink kits between 0 and 4 wk of
age and 0.5, 5, 25, and 50 mmol/l for animals 6 wk and older. Because
of the limited number of tissues that could be prepared from the short
intestine, rates of amino acid uptake by the midintestine were measured
at only three concentrations (tracer alone and in the presence of 25 and 50 mmol/l unlabeled amino acid). The selection of 25 and 50 mmol/l
concentrations of amino acid was based on Michaelis constant
(Km) of 1-10 mmol/l that we and others have
measured using the same method to study other mammals (15, 16; our
unpublished data for pigs). Therefore, it was speculated that
if the amino acid transporters of mink are similar to those of other
mammals, 25 mmol/l should be sufficiently high to saturate the
carriers, and the slopes of the lines between 25 and 50 mmol/l would
approximate the carrier-independent influx for the amino acids.
Osmolarity of all nutrient solutions was maintained at 290 mosmol/l by an isosmotic reduction of the NaCl (maximum
reduction of 25 of the 117 mmol/l), and pH was 7.4 when aerated with
the gas mixture. It was necessary to adjust the pH of the aspartate solution by adding NaOH.
To determine whether a saturable component of absorption was present,
we compared accumulation of tracer sugar and amino acid when present
alone compared with when added to 50 mmol/l of unlabeled sugar and
amino acid (accumulation ratios). We assumed that if transporters were
present in limited numbers (a saturable component is present), the
presence of 50 mmol/l unlabeled sugar or amino acid would reduce the
accumulation of tracer due to competition. As a consequence,
accumulation ratios greater than a value of 1.0 were considered to
indicate the presence of a saturable component. In contrast, values not
different from 1.0 would indicate that tracer influx is independent of
the concentration of unlabeled nutrient and that absorption is largely
by simple diffusion. This could occur if the transporters are absent or
are present in very low densities.
BBMV studies.
BBMV were prepared using a standard protocol (26).
Accumulation of tracer by the BBMV was measured using a fast-sampling, rapid filtration device programmed to collect nine samples over the
first 2.7 s of incubation for methionine, leucine, and
glycyl-sarcosine; 3.6 s for aspartate, and 4.5 s for proline,
lysine, and glucose. The different time periods were selected so that
accumulation could be measured during the linear phase. Accumulation of
tracer was studied in the presence of 0 and 200 mmol/l NaCl to
determine whether uptake is sodium dependent and with 200 mmol/l NaCl
and 100 mmol/l unlabeled nutrient to determine whether there is
competition for transporter sites. Osmolarity of internal and external
solutions was maintained constant by varying mannitol concentrations.
Initial rates of BBMV uptake were calculated by linear regression
analysis. When a curve deviated from linearity, the initial rate was
estimated from the first-degree coefficient of the second-degree polynomial.
Chemicals
All reagents used to prepare solutions were purchased from
Sigma (St. Louis, MO) and were of the highest purity available. Radiolabeled compounds were purchased from New England Nuclear (Amherst, MA: D-[14C]glucose,
L-[3H]glucose,
D-[14C]fructose,
[14C]polyethylene glycol, and Mississaugua, ON, Canada:
D-[1-3H]glucose) or Amersham (Oakville, ON,
Canada; glycyl [N-methyl-3H]sarcosine,
L-[2,3,4,5-3H]arginine,
L-[methyl-3H]methionine,
L-[4,5-3H]leucine,
L-[4,5-3H]lysine,
D-[2,3-3H]aspartic acid, and
L-[2,3,4,5-3H]proline).
Data Analysis and Statistics
Values presented in the text, tables, and figures are means ± SE. The main effects of intestinal region and age on rates of absorption by intact tissues were evaluated using the PROC GLM procedure of SAS (Statistical Analysis Systems, version 6.11). When a
significant effect was detected, specific differences were identified
by Duncan's test. The PROC Univariate procedure of SAS was used to
determine whether the accumulation ratios exceeded a value of 1.0. If
so, this was considered to be indicative of competition between the
tracer and unlabeled amino acid for a limited number of transporter
sites, and that a portion of absorption was via a saturable pathway.
For all analyses, a value of P < 0.05 was accepted as
the critical level of significance.
Kinetics of sugar transport by intact tissues were defined using the
Enzfitter nonlinear regression analysis program (Biosoft, Elsevier,
UK). Glucose data were fit to model equations that included one and two
transporters to estimate maximum rates of absorption (Vmax) and apparent affinity constants
(Km*). The model equation providing
the best fit of the data (based on examination of the residuals) was
used for estimating the kinetic parameters. Fructose data were
initially fit to a linear regression and then to a model equation for a
single transporter to determine whether this improved the fit. The
limited number of concentrations used to study amino acid uptake
precluded kinetic analysis.
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RESULTS |
Body Weights and Intestinal Dimensions
Age influences.
Mean body mass increased from 11 ± 0.4 g at birth to
367 ± 17 g at 6 wk when the kits were weaned and was
719 ± 36 g at 8 wk. The adult females weighed 1,183 ± 101 g. Intestinal length increased from birth to 8 wk of age, but
did not increase between 8 wk and maturity (Fig.
1A), despite the 65% increase
in body mass. Intestinal length normalized to body mass (cm/kg)
declined during development (P < 0.05). Double-log
plots of intestinal length and body mass (data not presented) revealed
a highly significant correlation (r2 = 0.97; P < 0.0001) with a slope of 0.41 ± 0.008. This is greater than the value of 0.33 predicted from dimensional
analysis (P < 0.05) (17).
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Fig. 1.
Intestinal dimensions of mink from birth to maturity.
Values for length (A) and mass (B) are expressed
as centimeters and grams and normalized to body mass. Values with
different letters are significantly different (P < 0.05), with lowercase letters used for comparisons of absolute lengths
and mass and capital letters used for comparisons of values normalized
to body mass.
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Total intestinal mass (estimated by multiplying the average for weights
of the 1-cm sleeves from the different regions times the length of the
intestine) increased between birth and 8 wk and then actually declined
20% between 8 wk and maturity (Fig. 1B). Intestinal mass
normalized to whole body mass (g/kg), doubled during the first 24 h after birth, remained stable for the next 2 wk, was higher at 4 and 6 wk, and then began to decline with the lowest values recorded from
mature animals. Double-log plots of body and intestinal mass revealed a
highly significant relationship (r2 = 0.96;
P < 0.0001) with a slope of 1.02 ± 0.02. This
value does not differ from the predicted value of 1.0 (17).
Sugar and Amino Acid Uptake
Age influences.
There was a significant effect of age on rates of uptake by intact
tissues (P < 0.01), with the patterns varying among
the different sugars and amino acids. Glucose uptake per milligram intestine at 50 mmol/l averaged from the different sites of small intestine (2 for mink 1 wk old and younger and 3 for mink 2 wk and
older) was highest at birth and declined during the first 24 h of
suckling and between consecutive age groups up to 4 wk (Fig.
2A; P < 0.05). Thereafter, rates of glucose uptake remained stable into
maturity. In contrast, initial rates of tracer D-glucose uptake by BBMV in the presence of an inwardly directed 200 mmol Na+ gradient increased during suckling (Fig.
2B).
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Fig. 2.
Rates of carrier-mediated glucose transport by intact
tissues (A) from birth to maturity for mink. Values are the
average for the proximal, mid, and distal small intestine, with
significant differences (P < 0.05) between age groups
indicated by different letters. Accumulation of tracer
D-glucose by brush-border membrane vesicles (BBMV) prepared
from the proximal and distal halves of the small intestine of mink from
2 wk to maturity (B).
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Fructose uptake by intact tissues did not change during the first
24 h after birth but had declined by 1 wk (Fig.
3; P < 0.05). Values
during suckling, adolescence, and maturity remained lower than those
during the neonatal period (0 days to 1 wk). Interestingly, fructose
uptake by the adult intestine was greater than at 8 wk (P < 0.05).
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Fig. 3.
Rates of carrier-mediated fructose transport by intact
tissues from birth to maturity for mink. Values are the average for the
proximal, mid, and distal small intestine, with significant differences
(P < 0.05) between age groups indicated by different
letters.
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Rates of amino acid uptake by intact tissues exposed to 50 mmol/l were
highest during the neonatal period and showed an increase during the
first 24 h after birth. Values declined thereafter, with the onset
of the decline varying among the four different classes of amino acids.
Compared with values at 24 h after birth, the decline was already
significant at 1 wk for aspartate, at 2 wk for proline (Fig.
4A), and at 4 wk for lysine,
leucine, and methionine (Figs.
5A-7A).
Rates of amino acid absorption at 8 wk and maturity were comparable to
those measured at 6 wk, indicating that weaning to the production diet
did not induce higher rates of absorption. Rates of amino acid
absorption by midintestine at tracer concentration and at 25 mmol/l
showed similar patterns of postnatal declines (P = 0.0004 and 0.0001) followed by stable values between 6 wk and maturity.
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Fig. 4.
Rates of total L-proline absorption (carrier
mediated and carrier independent) by intact tissues (A) from
birth to maturity for mink Values are the average for the proximal,
mid, and distal small intestine, with significant differences between
age groups indicated by different letters. Accumulation of tracer
L-proline by BBMV prepared from the proximal (B)
and distal (C) halves of the small intestine from 2 wk to
maturity. BBMV proline accumulation was measured with an inwardly
directed 200 mmol/l Na+ gradient (first bar), 0 mmol
external Na+ (second bar), and in the presence of an
external solution containing 200 mmol/l Na+ and 100 mmol/l
unlabeled proline (third bar).
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Fig. 5.
Rates of total L-lysine absorption (carrier
mediated and carrier independent) by intact tissues (A) from
birth to maturity for mink. Values are the average for the proximal,
mid, and distal small intestine, with significant differences between
age groups indicated by different letters. Accumulation of tracer
L-lysine by BBMV prepared from the proximal (B)
and distal (C) halves of the small intestine from 2 wk to
maturity. BBMV lysine accumulation was measured with an inwardly
directed 200 mmol/l Na+ gradient (first bar), 0 mmol
external Na+ (second bar), and in the presence of an
external solution containing 200 mmol/l Na+ and 100 mmol/l
unlabeled lysine (third bar).
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Initial rates of BBMV uptake measured in the presence of a 200 mmol/l
inwardly directed Na+ gradient also varied among the
different ages for each amino acid. Peak tracer accumulation was
recorded at 4 weeks for aspartate (Fig.
8, B and C), at 6 weeks for methionine (Fig. 7, B and C), and after
weaning for lysine and leucine (Figs. 5, B and C
and 6, B and C). BBMV proline uptake rates
increased between birth and maturity (Fig. 4, B and
C), with the exception of the high rates measured in the
distal small intestine at 4 wk.
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Fig. 6.
Rates of total L-leucine absorption (carrier
mediated and carrier independent) by intact tissues (A) from
birth to maturity for mink. Values are the average for the proximal,
mid, and distal small intestine, with significant differences between
age groups indicated by different letters. Accumulation of tracer
L-leucine by BBMV prepared from the proximal (B)
and distal (C) halves of the small intestine from 2 wk to
maturity. BBMV leucine accumulation was measured with an inwardly
directed 200 mmol/l Na+ gradient (first bar), 0 mmol
external Na+ (second bar), and in the presence of an
external solution containing 200 mmol/l Na+ and 100 mmol/l
unlabeled leucine (third bar).
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BBMV uptake of the dipeptide glycyl-sarcosine did not differ
significantly among ages for the proximal small intestine but increased
between 2 and 8 wk in the distal intestine (Fig.
9).
Regional distribution.
A significant declining proximal-to-distal gradient of glucose uptake
by intact tissues was present only during suckling (birth to 4 wk) with
values in the proximal intestine averaging 1.9-fold higher (±0.3) than
in the distal intestine (data not shown). From 6 wk into maturity,
regional differences were not detected. The BBMV data (Fig.
2, B and C) provided a contrasting
scenario in that from 2 to 8 wk initial rates of glucose uptake by BBMV
prepared from proximal intestine averaged ~60% of values measured
for distal intestine (P < 0.05). Between 8 wk and
maturity there was a redistribution of BBMV glucose uptake resulting in
rates by the proximal intestine exceeding those for the distal
intestine by 2.3-fold (P < 0.05).
Rates of fructose uptake by intact tissues did not differ between
regions at birth and 24 h and from week 6 to maturity.
However, from week 1 to 4 there was a declining
proximal-to-distal gradient for rates of fructose uptake.
A redistribution of intact tissue absorption along the length of the
small intestine was detected for all five amino acids. At birth and
during early neonatal development (up to 1 wk), rates of absorption
averaged for all five amino acids did not differ between the two
segments studied. During the remainder of suckling (weeks
2-6), rates of amino acid absorption declined from the proximal to distal regions. Before or at the time of weaning, there was
a shift such that at 8 wk and in maturity, rates of absorption were
higher in the distal intestine compared with the proximal segment.
Rates of tracer accumulation by BBMV did not differ between proximal
and distal intestine at any age for aspartate (Fig. 8, B and
C) and glycyl-sarcosine (Fig. 9, B and
C), whereas lysine uptake was higher in distal intestine at
6 and 8 wk (Fig. 5, B and C; P < 0.05). In contrast, BBMV leucine uptake was higher in the proximal
intestine at 6 wk and maturity (Fig. 6, B and C;
P < 0.05) but was higher for the distal intestine at
all other ages. Rates of BBMV methionine uptake were higher in the
proximal segment at all ages, except maturity (Fig. 7, B and
C). BBMV proline uptake shifted from being higher in the
distal intestine at 2 and 4 wk to becoming higher in the proximal
intestine at 6 wk and older (Fig. 4, B and C).
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Fig. 7.
Rates of total L-methionine absorption
(carrier mediated and carrier independent) by intact tissues
(A) from birth to maturity for mink. Values are the average
for the proximal, mid, and distal small intestine, with significant
differences between age groups indicated by different letters.
Accumulation of tracer L-methionine by BBMV prepared from
the proximal (B) and distal (C) halves of the
small intestine from 2 wk to maturity. BBMV methionine accumulation was
measured with an inwardly directed 200 mmol/l Na+ gradient
(first bar), 0 mmol external Na+ (second bar), and in the
presence of an external solution containing 200 mmol/l Na+
and 100 mmol/l unlabeled methionine (third bar).
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Kinetics of sugar uptake.
During the first week after birth, accumulation ratios for
D-glucose uptake by intact tissue of the proximal intestine
averaged 111-fold (±17) (P < 0.01). The accumulation
ratios declined during the remainder of suckling (data not shown), but
from 6 wk into maturity the ratios were relatively stable and averaged
6.2 ± 0.4. These data suggest that throughout development, tracer
and unlabeled D-glucose compete for a limited number of
transporters and provide evidence for a saturable component of
carrier-mediated D-glucose uptake.
At all ages, the concentration-uptake data for glucose did not show
obvious saturation kinetics (presence of distinct plateau region) and
best fit a model equation for two transporters (Table 1). A high-affinity system
(Km* averaged 0.55 ± 0.18 mmol/l for all ages) was evident during suckling (birth to 4 wk), but Vmax values were negligible during and
after weaning (from week 6 to maturity). The second system was characterized by a lower affinity (58 ± 21 mmol/l for all ages), which made it difficult to accurately define age-related changes
in kinetic characteristics based on the concentrations used.
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Table 1.
Kinetic constants for the carrier-mediated transport of glucose and
fructose by the proximal intestine of mink from birth to maturity.
Selected equations represent the lowest order equations that
provided the best fit based on residuals
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Fructose accumulation ratios for intact tissues averaged 2.8 ± 0.3 for the first 2 wk after birth. During this period, the uptake-concentration data best fit a model for a single, low-affinity transport system (Km* averaged 68 ± 32 mmol/l). For the remainder of suckling and at 8 wk, accumulation ratios
averaged 0.6 ± 0.1. This value does not exceed 1.0, indicating
there is a lack of competition between tracer and unlabeled fructose
for carriers. Corresponding with these findings, fitting the
uptake-concentration data for 4, 6, and 8 wk to an equation with a
saturable component did not improve the fit over that obtained for
linear, nonsaturable uptake. Accumulation ratios for adults (1.5 ± 0.2) were >1.0 (P < 0.05), and analysis of the
uptake-concentration data suggested the presence of a carrier with a
Km* of 24 mmol/l.
Characteristics of amino acid transport by intact tissues and BBMV.
Accumulation ratios for intact tissues from the midintestine exceeded
1.0 for all amino acids during early development (from birth to 2 wk;
P < 0.05) and for adults (P < 0.05).
The ratios were lower at 4, 6, and 8 wk, and during this period ratios
did not differ from 1.0 for lysine and proline. BBMV accumulation of
tracer aspartate, leucine, methionine, and proline was inhibited in the
presence of 100 mmol/l of the corresponding unlabeled form at all ages
and in both proximal and distal halves of the small intestine (Figs. 4,
6, 7, 8; B and C). Accumulation of tracer lysine
by both proximal and distal intestine was only partially inhibited by
100 mmol/l unlabeled lysine (Fig. 5, B and C),
with similar findings for glycyl-sarcosine (Fig. 9). Collectively, the
findings from the intact tissues and BBMV suggest low-affinity amino
acid carriers (not saturated at 50 mmol/l) may be present and that the
increase in rates of absorption between 25 and 50 mmol/l may not be
indicative of carrier-independent, diffusive influx.
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Fig. 8.
Rates of total L-aspartate absorption
(carrier mediated and carrier independent) by intact tissues
(A) from birth to maturity for mink. Values are the average
for the proximal, mid, and distal small intestine, with significant
differences (P < 0.05) between age groups indicated by
different letters. Accumulation of tracer L-aspartate by
BBMV prepared from the proximal (B) and distal
(C) halves of the small intestine from 2 wk to maturity.
BBMV aspartate accumulation was measured with an inwardly directed 200 mmol/l Na+ gradient (first bar), 0 mmol external
Na+ (second bar), and in the presence of an external
solution containing 200 mmol/l Na+ and 100 mmol/l unlabeled
aspartate (third bar).
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Fig. 9.
Accumulation of tracer glycyl-sarcosine by BBMV prepared
from the proximal (A) and distal (B) halves of
the small intestine mink from 2 wk to maturity. BBMV glycyl-sarcosine
accumulation was measured with an inwardly directed 200 mmol/l
Na+ gradient (first bar), 0 mmol external Na+
(second bar), and in the presence of an external solution containing
200 mmol/l Na+ and 100 mmol/l unlabeled glycyl-sarcosine
(third bar).
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BBMV uptake of tracer aspartate and proline was virtually entirely
Na+ dependent in both proximal and distal small intestine
from 2 wk to maturity, whereas significant proportions of lysine,
leucine, and methionine uptake were not Na+ dependent.
Accumulation of tracer glycyl-sarcosine did not differ when measured in
the presence or absence of an inwardly directed Na+
gradient (Fig. 9), with the exception of the proximal intestine of
adult mink; Na+ dependency was not determined in the distal
intestine of adult mink.
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DISCUSSION |
Intestinal Dimensions
The intestines of mink are relatively short throughout ontogeny
(10) compared with those omnivorous mammals, which is
typical for carnivores (22). However, when normalized to
body mass, the intestines of adult mink (123 cm/kg) are longer than
those of 3.6-kg adult cats (59 cm/kg; P < 0.05) and
12.8-kg adult dogs (71 cm/kg: P < 0.05)
(8). Despite this, the transit times for food in mink
(2, 14) are more rapid than in cats and dogs (8).
For some species, including the mink (present study), the first
swallows of colostrum after birth trigger intestinal growth and changes
in intestinal functions (25, 26). The neonatal responses
are not universal, as evident from the lack of intestinal growth of
kittens during the first week after birth (7, 8). More is
known about the weaning period during the transition from a milk diet
to a solid diet when there are increases in intestinal dimensions of
rats (23), pigs (18), cats (7,
8), and other mammals (3). In contrast to these
species, mink do not experience a similar postweaning increase in
intestinal size, despite a continuing increase in body size. In fact,
intestinal mass of mink actually declines between 8 wk and maturity.
Absorption of Amino Acids and Sugars
Ontogenetic changes in intestinal structure and functions are
considered to be set by genetic determinants to match anticipated shifts in diet composition and the requirements for energy and nutrients (5). From our findings for the cat
(7), the a priori expectation was that during postnatal
development of mink, rates of uptake would decline for sugars, whereas
absorption of amino acids would increase to match the shift from a milk
diet to an adult diet low in carbohydrate and high in protein. The most
dramatic changes were expected to occur at birth and again at weaning,
when changes in the rates of enterocyte proliferation and replacement
alter villus and crypts dimensions and are thought to affect intestinal
functions, such as nutrient transport (12) and lactase
activity (9).
Sugars.
The kinetic data from the intact tissues suggest that throughout
postnatal development, glucose is absorbed by two systems that differ
in affinity and capacities. These findings contrast with the single
high-affinity system for glucose transport detected in postnatal
omnivores (18, 23, 26). The declines in maximum rates of
uptake for both systems and for rates of uptake at 50 mmol/l leading up
to weaning are consistent with decreases in the densities of
transporters. The lack of change between 6 wk (weaning) and maturity
for rates of intact tissue glucose uptake at 50 mmol/l differs from the
postweaning decline detected in cats (7, 8), and in
several ways is more similar to the pattern reported for the rat.
Moreover, the age-related increases in initial rates of BBMV tracer
glucose uptake particularly in the proximal intestine are compatible
with a shift to a greater proportion of uptake via the high capacity
system. When the increases in intestinal weight per centimeter during
development are considered, rates of glucose uptake normalized to
length or surface area actually increase after weaning. Collectively,
these findings suggest that the total number of glucose transporters
per unit of intestine actually increase after weaning instead of
decline, as would be expected from the decrease in dietary carbohydrates.
Age-related changes in fructose uptake by intact tissues are even more
paradoxical because it is absent or present only in negligible amounts
in milk and the natural and production diets consumed by adult wild and
domestic mink. However, rates of transport were highest during the
neonatal period (birth to 2 wk), when accumulation ratios exceeded 1.0, and it was possible to resolve a low-affinity transport system. The
nearly twofold increase in fructose uptake between 8 wk and maturity is
similar to the pattern seen in the rat (23), but very
different from the stable values seen in the cat (7, 8),
and suggests a reappearance of the low-affinity carrier system.
Expressing rates of fructose uptake relative to those for glucose (F/G
ratios) provides additional insights into age-related shifts in the
abilities of mink to absorb sugars. Rates of glucose uptake exceed
those for fructose throughout postnatal life of the mink with F/G
ratios ranging from 0.21 at birth to 0.62 at maturity. These values are
comparable to those for rats (23) and rabbits
(6) but are higher than those for cats, which are stable
during development at 0.14 (7, 8). It is interesting that
the increase in F/G ratios from 0.26 ± 0.02 at 2 wk to 0.51 ± 0.05 at 4 wk (P < 0.05) is similar to the pattern
for rats and coincides with when both species shift to the adult diet.
Amino acids.
Unlike the significant postweaning declines in rates of amino acid
absorption by intact tissues of rats and cats, values for the mink
intestine remained relatively constant after 6 wk. The different
patterns among species may be partly explained by the composition of
the adult diet. Specifically, rats are weaned to commercial diets that
are comparatively low in protein (20-25%) and correspondingly
exhibit greater proportional declines in rates of amino acid
absorption. Not only is the protein content of commercial diets fed to
cats (~45%) lower than that of the production diet fed to mink
(56%), the plant-based proteins often added to diets fed to cats
(e.g., corn gluten) have lower digestibility, hence reducing the
concentrations of amino acids and peptides available for intestinal
absorption. Despite the high dietary loads of digestible protein
consumed by adult mink, rates of amino acid absorption at 50 mmol/l
(sum of both carrier-mediated and carrier-independent pathways)
normalized to tissue mass were not different from those for weaned rats
(23), cats (7), and dogs (8).
This contrasts with our speculation that rates of amino acid and
peptide absorption would be higher in weaned mink than other species to
allow for the rapid and efficient digestion of the high dietary loads
of protein.
The characteristics of amino acid absorption are not as well understood
as for sugar transport, and most of what is known is for omnivores. The
BBMV data indicate that aspartate and proline are transported by a
saturable process that is almost entirely Na+ dependent,
whereas a Na+-independent pathway is present for
accumulation of leucine, methionine, and lysine. The low accumulation
ratios for aspartate, leucine, methionine, and proline based on intact
tissues, despite inhibition of initial rates of BBMV uptake by 100 mmol/l unlabeled amino acids could result if the amino acid
transporters of mink exist at such high densities that 50 mmol/l is not
high enough to cause saturation. However, rates of accumulation of
amino acids by BBMV from adults were lower than the rates for glucose.
Alternatively and teleologically more reasonable, the amino acid and
peptide transporters of mink may have low affinities for their
respective substrates, but high capacities for transport, and this may
be particularly true for lysine with BBMV accumulation not fully inhibited by 100 mmol/l. The presence of low-affinity systems would
allow a fewer number of transporters to rapidly and efficiently absorb
the high dietary loads of amino acids, but this needs to be verified.
Capacities.
The capacities of the entire length of small intestine to absorb
glucose and fructose increased from birth to adulthood (Fig. 10), particularly for fructose, and did
not scale directly to intestinal dimensions, which would be the case if
the majority of uptake was via a paracellular pathway
(17). Furthermore, the continuing increases in
capacities after 8 wk, despite declines in intestinal mass, provide
further evidence that the densities of sugar transporters per unit mass
increased. The increase in capacities to transport fructose during the
first 24 h, but not for glucose, is consistent with the presence
in the apical membrane of at least two different transport systems for
sugars.
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Fig. 10.
Uptake capacities for the entire length of small
intestine of mink from birth to maturity for sugars (A) and
amino acids (B). Values are calculated by integrating rates
of uptake by intact tissues (nmol/mg-min) with total mass of small
intestine.
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Amino acid absorption capacities increased twofold or more between
birth and 24 h. Slopes for double log plots of absorption capacities versus whole body mass revealed increases between 1 day and
8 wk that were similar to the value of 0.75 predicted from dimensional
analysis (17). Interestingly and in sharp contrast to the
sugars, the capacities to absorb amino acids declined between 8 wk and
maturity, despite increases in whole body mass.
Perspectives
There is a need to better understand the types and characteristics
of nutrient transporters present in the apical membrane of the small
intestine and how they are regulated by dietary loads (quantities and
composition). Our findings suggest the ability of the short intestine
of mink to rapidly and efficiently process large dietary loads of
protein is partly dependent on the presence of multiple amino acid
transport systems that have different affinities and Na+
dependency. However, rates of amino acid absorption at 50 mmol/l by
intact tissues are not markedly higher for the mink compared with
values we and others have measured in omnivores and herbivores using
the same methods (6, 16, 23; our unpublished data for pigs). Moreover,
dipeptide absorption does not appear to provide a major mechanism of
absorption because glycyl-sarcosine accumulation by BBMV was lower than
the uptake of any of the amino acids. Kinetic studies are needed to
elucidate the functional characteristics and relative roles of the
various amino acid and peptide transport systems at different stages of
development and to search for alternative pathways for absorbing the
products of protein hydrolysis. Also to be considered, but not well
understood, are the concentrations of substrates for the respective
carriers that actually exist at the apical membrane.
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ACKNOWLEDGEMENTS |
We thank Merete Stubgård for assisting in studies performed in
Denmark, Claudie Leroy for technical support for the BBMV studies conducted at the University of Montreal, and Dr. Stephen Secor for
reviewing the manuscript.
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FOOTNOTES |
The Danish Fur Breeders Association, the Danish Agricultural and
Veterinary Research Council, National Science and Engineering Research
Council of Canada, and Mississippi State University provided financial support.
Address for reprint requests and other correspondence: R. K. Buddington, Dept. of Biological Sciences, Mississippi State Univ., Mississippi State, Mississippi 39762 (E-mail: rkb1{at}ra.msstate.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 May 2000; accepted in final form 8 August 2000.
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ABSTRACT
INTRODUCTION
