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First published online October 31, 2008
Journal of Experimental Biology 211, 3544-3553 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.020644
Mechanism and rate of glucose absorption differ between an Australian honeyeater (Meliphagidae) and a lorikeet (Loriidae)
School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia
* Author for correspondence (e-mail: K.Napier{at}murdoch.edu.au)
Accepted 22 September 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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0%). Over time, however, more glucose may be absorbed via the
paracellular route. Glucose absorption by both mediated and non-mediated
mechanisms in wattlebirds occurred at a faster rate than in lorikeets, and
wattlebirds also rely substantially on paracellular uptake. In wattlebirds, we
recorded higher bioavailability of L-glucose (96±3%)
compared with D-glucose (57±2%), suggesting problems with
the in vivo use of radiolabeled D-glucose. Further trials
with 3-O-methyl-D-glucose revealed high bioavailability in
wattlebirds (90±5%). This non-metabolisable glucose analogue remains
the probe of choice for measuring uptake rates in vivo, especially in
birds in which absorption and metabolism occur extremely rapidly.
Key words: paracellular absorption, glucose absorption, red wattlebird, Anthochaera carunculata, rainbow lorikeet, Trichoglossus haematodus, 3-O-methyl-D-glucose, L-glucose
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Karasov,
1990; Karasov and Hume,
1997). In non-flying mammals, total carrier-mediated absorptive
capacity, usually extrapolated from in vitro nutrient uptake
measurements by the everted sleeve technique
(Karasov and Diamond, 1983),
generally either meets or exceeds the daily dietary carbohydrate intake of the
animal (Ferraris and Diamond,
1989). However, in many avian species, capacity for mediated
glucose transport extrapolated from in vitro measurements is
significantly lower than observed total in vivo glucose assimilation
(Caviedes-Vidal and Karasov,
1996; Karasov and Cork,
1994; Levey and Cipollini,
1996; McWhorter et al.,
2006). How, then, do small volant vertebrates maintain high
digestive efficiency?
Water-soluble nutrients such as carbohydrates and amino acids are absorbed
in the small intestine by both protein-carrier-mediated (transcellular) and
non-mediated (paracellular) mechanisms
(Hopfer, 1987). The
paracellular pathway is located between adjacent epithelial cells, where the
tight junctions (zonula occludens) join the cells, block the movement of
integral membrane proteins between the apical and basolateral membranes, and
constrain the movement of water and hydrophilic solutes across the junction
(Anderson, 2001;
Ballard et al., 1995;
McWhorter, 2005;
van Itallie and Anderson,
2006). Tight junction permeability is variable and appears to be
regulated by physiological mechanisms
(Mitic et al., 2000;
Pappenheimer, 1987;
Powell, 1981;
Turner, 2000). Pappenheimer
(Pappenheimer, 1993) suggested
that paracellular nutrient absorption may offer a selective advantage because
it provides non-saturating absorptive capacity that is matched to the rate of
substrate hydrolysis in the intestine and it requires little energy. High
fractional absorption, or bioavailability, of small non-transported,
metabolically inert, water soluble probe molecules (e.g. stereoisomers of
simple sugars) has often been taken as evidence of significant paracellular
nutrient absorption in small volant vertebrates
(Caviedes-Vidal et al., 2007;
Chang and Karasov, 2004;
McWhorter, 2005;
McWhorter et al., 2006). The
nutritional importance of paracellular absorption was a subject of
considerable debate in the past, however, partly because differences in
methodology made comparisons across studies problematic
(McWhorter, 2005). More recent
studies employing uniform methods have shown convincingly that small birds and
bats rely more on non-mediated mechanisms of absorption than do non-flying
mammals, perhaps as a compensation for smaller intestines
(Caviedes-Vidal et al., 2007;
Tracy et al., 2007).
A criticism that has been levelled at studies using only bioavailability
data to draw conclusions about the nutritional importance of paracellular
uptake is that probe compounds might be absorbed at a much slower rate than
nutrients absorbed by mediated mechanisms, but over the entire length of the
intestine and extended time of digesta residence in the gut
(Schwartz et al., 1995). In
this case, probe fractional absorption may be high, falsely indicating that
paracellular absorption accounts for a high proportion of total nutrient
absorption. An elegant approach to resolving this issue, and the one which we
have adopted here, is to simultaneously compare the extent and rate of
absorption of passively and actively absorbed probe molecules in
vivo. 3-O-methyl-D-glucose (hereafter 3-OMG) is a
non-metabolisable D-glucose analogue that competes for the same
co-transporters as D-glucose
(Solberg and Diamond, 1987)
and has a similar maximal transport rate to D-glucose in mammals
(Thomson et al., 1982).
Comparing the relative rates of absorption of 3-OMG (absorbed via
both mediated and non-mediated mechanisms) and L-glucose [absorbed
by non-mediated mechanisms, recently verified in birds by competitive
inhibition studies (Chang et al.,
2004)] in granivorous house sparrows (Passer domesticus),
Chang and Karasov (Chang and Karasov,
2004) estimated that at least 70% of total glucose uptake was
paracellular. More recently, radiolabeled D-glucose (absorbed
via both mediated and non-mediated mechanisms and catabolised after
absorption) was tested as a probe for studying the kinetics of glucose
absorption in vivo, in the seasonally frugivorous American robin
(Turdus migratorius Turdidae)
(McWhorter et al., 2008).
These authors found that the kinetics of absorption and elimination of
D-glucose appear to be suitable for such in vivo studies,
and measurements using absorption of D-glucose and 3-OMG in this
species give comparable estimates of the relative contribution of paracellular
absorption [60±8 vs 62±1% (means ± s.e.m.) of
total glucose uptake averaged over the first 20 min of absorption].
To date, little data have been collected on the in vivo kinetics
of glucose absorption by nectarivorous birds. These birds may be particularly
interesting as models of digestive mechanisms, since they tend to have
significantly shorter alimentary tracts compared with insectivorous species of
the same body mass (Richardson and
Wooller, 1986). Karasov and Cork
(Karasov and Cork, 1994)
studied paracellular absorption in the nectarivorous/frugivorous rainbow
lorikeet (Trichoglossus haematodus, Loriidae) by measuring the
bioavailability of L-glucose. They concluded, based only on extent
of L-glucose absorption (80%), that these lorikeets rely
significantly upon paracellular absorption for glucose uptake. In the present
study, we revisited glucose absorption in the rainbow lorikeet and compared
this species to a large Australian honeyeater, the red wattlebird
(Anthochaera carunculata, Meliphagidae).
Aims of the present study
Our first aim was to further assess radiolabeled D-glucose as a
probe for in vivo pharmacokinetic studies of glucose absorption. We
predicted that radiolabeled D-glucose would prove suitable for such
studies. We assessed the kinetics of elimination of
D-[14C]glucose in red wattlebirds and rainbow lorikeets
after intramuscular injection, and of absorption after oral administration. We
compared our pharmacokinetic results with apparent assimilation efficiency
(AE*) of non-radiolabeled D-glucose measured by a
traditional 24 h mass balance protocol. Because these values were vastly
different in red wattlebirds, we repeated the pharmacokinetic trials in this
species using [14C]3-OMG, a glucose analogue commonly used for
in vivo glucose absorption studies
(Chang and Karasov, 2004;
Tracy et al., 2007). We also
tracked the movement of D-[14C]glucose (and
L-[3H]glucose, see below) into the various tissues of
both study species over time to assess whether the bioavailability of
D-glucose as calculated by our pharmacokinetic methods was
erroneously low because of rapid post-absorption catabolism or sequestration
in tissues (especially gut tissues and liver).
Our second aim was to estimate the proportional contribution of
paracellular to total glucose uptake in our study species. We predicted that
paracellular absorption would account for the majority of total glucose
absorption in both of these nectarivorous birds. We compared the absorption
kinetics of simultaneously administered D-[14C]glucose
and L-[3H]glucose. The ratio of relative rates of
absorption (or, alternately, the cumulative fraction of an oral dose absorbed
at a given time point) of L-glucose to D-glucose or
3-OMG provides a robust estimate of the proportion of total glucose uptake
that is non-mediated (Chang and Karasov,
2004).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The birds were housed in individual cages (46 cmx56 cmx45 cm)
in a controlled environment room maintained at 21±2°C on a 12 h
automatic lighting regime with the photophase from 06.00 h to 18.00 h. During
the period of captivity the birds were fed a maintenance diet consisting of
Wombaroo® powder (main sugar type present in the form of sucrose; Wombaroo
Food Products, Adelaide, SA, Australia) supplemented with additional sucrose
(25% w/w of total dry matter). Both species received water ad
libitum, and rainbow lorikeets also periodically received assorted fresh
fruits such as apples and oranges.
For experiments, the birds were housed in individual experimental cages made from 3 mm thick opaque white polyvinyl chloride (PVC) plastic and clear acrylic sheeting (42 cmx54 cmx50 cm), with an automatic lighting regime with photophase from 07.00 h to 18.00 h. The acrylic front of the cage was coated with reflective Mylar® film to act as a one way mirror (cages lit inside, held in a darkened room) so birds would not be disturbed by outside movement. Red wattlebirds received the maintenance diet by accessing inverted stoppered syringes fixed to the outside of the back wall of the cage through a small opening cut into the cage, whereas rainbow lorikeets fed from commercially available plastic parrot feeders fixed to the inside of the back wall of the cage.
Measurement of L-glucose and D-glucose absorption in vivo
Food was removed 20 min prior to the experiments and measurements began
approximately 2.5 h after lights on. A cocktail containing either
D-[14C]glucose (rainbow lorikeets and red wattlebirds,
trial A) or [14C]3-OMG (red wattlebirds only, trial B) and
L-[3H]glucose (American Radiolabeled Chemicals, Saint
Louis, MO, USA) was administered orally (by gavage) and by intramuscular
injection (i.m.) to each bird in separate experiments (see
Table 1 for solution
compositions). Hereafter these radiolabeled compounds will be referred to
simply as D-glucose, 3-OMG and L-glucose. Trials were
separated by at least 2 weeks to ensure complete recovery from the process of
repeated blood collection and elimination of any residual radioactivity. The
order of trials and the sequence of treatment type given to the birds were
both randomly assigned. The volume of solution administered was measured by
weighing the syringe (±0.00001 g) before and after administration.
Aliquot samples of the oral and i.m. solutions were saved for radioactivity
analysis.
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An 50 µl blood sample was collected from the brachial vein prior to
each trial (time zero, t0) for background correction.
Following ingestion or injection of the probe solution, eight or nine brachial
vein blood samples were similarly collected at times (ti):
2.5 (trial B only), 5, 10, 20, 30, 45, 60, 120 and 240 min. After the first
hour of blood sampling, during which the birds were periodically offered
maintenance diet from a feeder while held in the hand, birds were transferred
to an experimental cage and received a maintenance diet solution ad
libitum. Immediately after blood sample collection, microcapillary tubes
were sealed with ChaSealTM clay tube sealing compound (Chase Scientific
Glass, Rockwood, TN, USA) and centrifuged in a microhematocrit centrifuge for
2–3 min at 9000g. Plasma samples and probe solution
aliquots were weighed (±0.00001 g), mixed with 3 ml of Ecolite+TM
liquid scintillation fluid (MP Biomedicals Australasia, Seven Hills, NSW,
Australia) and counted in a scintillation spectrometer (Beckman LS6500 Liquid
Scintillation Counter, Beckman Coulter, Fullerton, CA, USA) as disintegrations
per minute (d.p.m.). All counts were corrected for variable quenching and for
spill of 14C into the 3H channel.
Pharmacokinetic calculations
The level of radioactivity in each plasma sample (Ci)
was corrected by sample mass and plotted against actual sampling time,
ti. The extent of absorption of the radiolabeled probe was
estimated using the total area under the curve (AUCtotal), which
represents the total amount of radiolabeled probe absorbed from
t0 to time infinity (t
).
AUCtotal is calculated using the trapezoidal rule from
t0 to the last sampling point (tn) and
estimated assuming log-linear decline for the period from
tn to t as:
 | (1) |
; Gibaldi,
1991; Gibaldi and Perrier,
1982).
The fraction of the probe that reaches the systemic circulation is known as
the fractional absorption or systemic bioavailability of the probe
(Gibaldi, 1991). The
fractional absorption (f) of each probe was calculated by estimating
the ratio between the AUC for the plasma concentration–time curve for
oral administration (AUCoral) compared with the AUC of the i.m.
administration (AUCi.m.):
 | (2) |
).
Additional analyses relating to the time course and apparent rates of
absorption of probes were carried out using the Wagner–Nelson or the
Loo–Reigelman method as appropriate
(Gibaldi and Perrier, 1982;
Wagner and Nelson, 1963).
Parameters were derived for each individual bird by non-linear curve fitting
the plasma concentrations after i.m. administration of the probes at each time
point to the mono-exponential (one compartment,
C=C0e–kelt)
and bi-exponential (two compartment,
C=ae–
t+be–βt)
models by use of the Marquardt–Levenberg algorithm (SYSTAT Software,
SigmaPlot for Windows, San Jose, CA, USA)
(Marquardt, 1963). In 30 out
of 40 cases (covering L-glucose and D-glucose in both
study species), a bi-exponential model did not fit the elimination data
significantly better than a mono-exponential model by use of the
F-test (Fig. 1A,B,
insets), thus the assumption of a one-compartment model was deemed appropriate
for these probes (Motulsky and Ransnas,
1987). In all eight cases for 3-OMG (this probe was used in red
wattlebirds trial B only), a bi-exponential model fitted the data
significantly better than a mono-exponential model by use of the
F-test (Fig. 1C,
inset). Analyses relating to the apparent rate of absorption were thus carried
out under the assumption of an open two-compartment model and first order
elimination for 3-OMG.
|
 | (3) |
22°C) for 15 min with
500 µl of the glucose hexokinase–glucose-6-phosphate dehydrogenase
enzymatic assay reagent (Sigma Aldrich product code G3293, Castle Hill, NSW,
Australia). Absorbance was then measured at 340 nm by spectrophotometry (UV
mini 1240, Shimadzu Scientific Instruments, Balcatta, WA, Australia) relative
to distilled water.
Probe distribution in body tissues following oral administration
Six red wattlebirds and five rainbow lorikeets were weighed (±0.01
g) and gavaged with a cocktail containing D-[14C]glucose
and L-[3H]glucose (see
Table 1 for cocktail
composition). Individual birds were subjected to euthanasia by an overdose of
the inhalation anaesthetic Isoflurane at varying time points after gavage:
2.5, 5, 10, 20, 30 and 45 (red wattlebird only) minutes. Tissues were also
collected from one un-gavaged rainbow lorikeet for background radioactivity
levels and quench correction of tissues. A small blood sample was collected
from the heart with glass microcapillary tubes which were immediately sealed
with clay tube sealing compound and centrifuged in a microhematocrit
centrifuge for 2–3 min at 9000 g. The intestines
were excised from immediately proximal to the proventriculus to the cloaca and
sectioned into four (proventriculus, proximal, medial and distal sections) and
flushed clean with saline solution; the intestinal contents were collected
separately for each of the four sections. The whole kidneys, brain, liver and
as much as possible of the pectoralis muscle were collected, weighed and
frozen for later analysis. All organs and intestinal contents collected were
weighed (±0.00001 g) and samples of intestinal contents, plasma and
probe solution aliquots were mixed with 3 ml of Ecolite+TM liquid
scintillation fluid and the radioactivity counted in a scintillation
spectrometer.
A small sample of each of the organs (
200 mg) was weighed, solubilised
(SolvableTM, PerkinElmer, TH, Groningen, Netherlands) and decoloured with
30% H2O2 before being mixed with 15 ml of
Ecolite+TM liquid scintillation fluid and the radioactivity counted. Two
samples were processed for each organ, intestinal contents and plasma, and the
radioactivity in each sub-sample (d.p.m. mg–1) was averaged
and extrapolated to the whole organ mass to estimate the total radioactivity
present in each organ (d.p.m. organ–1). Plasma volume was
estimated from bird body mass in mg multiplied by 0.05, the approximate plasma
volume in birds (Goldstein and Skadhauge,
2000). The radioactivity of each organ was then expressed as a
percentage of total radioactivity delivered by oral gavage.
Statistical analysis
Numerical data are presented as mean ± s.e.m., with N
referring to the number of animals. Statistical analyses were performed on
data for individual birds. The validity of the normal distribution of
variables was tested by the one-sample Kolmogorov–Smirnov Test (SPSS,
SPSS, Inc, Chicago, IL, USA). Log-transformed elimination rate constants and
f values did not violate the assumptions of normality. Results were
analysed by repeated-measures ANOVA, one-way ANOVA, paired t-test and
independent samples t-test (SPSS) with degrees of freedom presented
as subscripts. Linear regression was by the method of least squares.
Statistical significance was accepted for <0.05.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Following simultaneous oral administration of D-glucose and L-glucose to rainbow lorikeets, average D-glucose concentration in plasma peaked by 20 min whereas L-glucose peaked later, at 45 min (Fig. 1D). D-glucose AUCoral in lorikeets was not significantly different to that of L-glucose (F1,7<0.01, P=0.988). In contrast to the injection trials in wattlebirds, D-glucose kel was significantly lower than that of L-glucose (F1,7=18.15, P=0.004) following oral administration, indicating slower elimination. The similar procedure for red wattlebirds (trial A) resulted in maximum average plasma D-glucose concentration at the first sampling time of 5 min, whereas plasma L-glucose peaked at 20 min; after these time points, both probes exhibited exponential decline (Fig. 1E). As in the lorikeets, D-glucose AUCoral in wattlebirds was not significantly different from that of L-glucose (F1,7=1.72, P=0.230). In agreement with the injection trial in red wattlebirds, D-glucose was eliminated more slowly than L-glucose, as indicated by a significant difference in kel values (F1,7=110.67, P<0.001).
Fractional absorption (f) of D-glucose significantly exceeded that of L-glucose in rainbow lorikeets; in red wattlebirds (trial A) there was also a significant difference between these probes, but D-glucose f was unexpectedly lower than that of L-glucose (Table 2). This surprising result led us to repeat the experiment using non-metabolisable 3-OMG in the red wattlebird (trial B), to remove the effects of pre-systemic catabolism of D-glucose. When red wattlebirds were gavaged with 3-OMG, average concentration in plasma peaked at 20 min after administration, and L-glucose peaked at 15 min (Fig. 1F), slightly earlier than in trial A. The observation that 3-OMG concentration in plasma peaked later that that of D-glucose (20 min vs 5 min; Fig. 1E,F) suggests a difference in the rate of absorption between the two D-glucose probes. As in the injection trial, 3-OMG was eliminated more slowly (kel was significantly lower) than L-glucose in red wattlebirds (F1,7=905.61, P<0.001).
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|
22.49,
P0.001, comparing both trial A and B). The ratio of the cumulative fractional absorption of L-glucose to D-glucose or 3-OMG at each sampling time point was used to estimate the proportion of total glucose absorption that occurs by non-mediated mechanisms (Fig. 2D–F). The L/D ratio increased with time after gavage and ranged from 0.10 to 0.97 (Fig. 2D) in rainbow lorikeets, indicating that at least 10% of D-glucose absorption occurred via the paracellular pathway. During the initial absorption phase from 5 min to 45 min in rainbow lorikeets, an average of 50% of the glucose was absorbed via the paracellular pathway. In red wattlebirds, the L/D ratio ranged from 1.12 to 2.06 (D-glucose; Fig. 2E) and 0.89 to 1.83 (3-OMG; Fig. 2F), indicating problems either with assumptions of this method of calculation, or with the probes themselves (see Discussion).
Apparent assimilation efficiency of D-glucose
Diet concentration did not significantly influence D-glucose
AE* in either rainbow lorikeets (F1,6=1.49,
P=0.270) or red wattlebirds (F1,6=0.001,
P=0.980), so the mean of all diet concentrations was calculated
(Table 2). D-glucose
AE* in rainbow lorikeets (99.7±0.1%) was not significantly
different from D-glucose bioavailability calculated using the
pharmacokinetic method (92.3±3.3%; F1,7=5.35,
P=0.054). For red wattlebirds, however, D-glucose
AE* by mass balance (99.8±0.1%) was significantly higher
than D-glucose bioavailability (f) estimated by the
pharmacokinetic method (57.4±2.4%; F1,7=326.71,
P<0.001), although the former was not significantly different from
3-OMG bioavailability in these birds (90.3±4.6%;
F1,14=4.21, P=0.059).
Probe distribution in body tissues following oral administration
The highest proportion of both D-glucose and
L-glucose recovered from the rainbow lorikeet (the sum of all
organs, extrapolated to the whole organ mass) peaked at our last time point of
30 min (Fig. 3E). This
contrasted with much faster recovery for red wattlebirds, with the highest
proportion of gavaged D-glucose and L-glucose recovered
peaking at our first sampling times (2.5 and 5 min;
Fig. 3E). These data provide
robust independent support for the conclusion that absorption of
D-glucose and L-glucose was slower in rainbow lorikeets
compared with red wattlebirds (above). However, the appearance of both probes
in various tissues and organs followed similar patterns over time for the two
species (Fig. 3) and from peak
values, we could project a path of radiolabeled glucose through tissues and
organs: gut contents
gut tissueplasma=liver=other
tissues/organs.
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Significantly more L-[3H]glucose, which should not
have been catabolised (Chang et al.,
2004; Karasov and Cork,
1994), was recovered from the intestinal contents of red
wattlebirds compared with rainbow lorikeets
(Fig. 3A;
t38=–2.34, P=0.025). There was no
significant difference in the recovery of L-[3H]glucose
between red wattlebirds and rainbow lorikeets from intestinal tissue
(Fig. 3B;
t37=–1.48, P=0.147), the liver
(Fig. 3D;
t9=–1.84, P=0.103) and other organs
(kidney, pectoralis and brain; t31=0.94, P=0.352;
Fig. 3E).
|
DISCUSSION |
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|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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In rainbow lorikeets, the bioavailability of D-glucose
calculated using our pharmacokinetic methodology was not significantly
different from apparent assimilation efficiency calculated using a traditional
mass-balance approach. As expected, the apparent rate of D-glucose
absorption (both mediated and non-mediated uptake) over the initial absorptive
period (through 45 min) significantly exceeded that of L-glucose
(non-mediated uptake only) (Chang et al.,
2004). Radiolabeled D-glucose thus appears to provide
robust in vivo estimates of absorption rates in this species. The
proportion of total glucose absorbed via the paracellular route
increased over time in rainbow lorikeets, with as little as 10% of total
glucose uptake via this non-mediated pathway at the first time point
recorded (Fig. 2D). The values
at the last two sampling time points (120 and 240 min, showing essentially
100% of total glucose absorption as paracellular) are probably indicative that
some L-glucose absorption is occurring further along in the
intestine, after D-glucose absorption was essentially complete
(Fig. 2D). L-Glucose
bioavailability measured in the present study (76%) compares favourably
with data collected by Karasov and Cork
(Karasov and Cork, 1994)
utilising a steady-state feeding methodology in these birds (80%).
L-Glucose bioavailability data, which do not provide any
information on relative rates of absorption, have been previously taken as an
estimate of the proportional contribution of paracellular uptake; our data
show that this contribution in rainbow lorikeets is both lower on average when
rates of absorption are considered, and varies through the absorptive
phase.
Although our data for the rainbow lorikeet were easily interpreted, the
same was not true of data for the highly nectarivorous
(Pyke, 1980) red wattlebird.
In these birds, estimated bioavailability of D-glucose using our
pharmacokinetic methodology was low (57%). We know this figure to be
incorrect, since the apparent assimilation efficiency of D-glucose
measured by mass balance was estimated at 99%. Furthermore, the
bioavailability of L-glucose (92–96%) was similarly high and
exceeded that of D-glucose, but based on mechanisms of absorption
we would expect that L-glucose absorption should at best match that
of D-glucose (Chang and Karasov,
2004). The absorption of L-glucose relative to
D-glucose (L/D ratio) in red wattlebirds
yielded values that exceeded 1 during the entire period of blood sampling
(Fig. 2E). This erroneous
result suggests that absorption rates based on the appearance of
14C in plasma after oral administration of radiolabeled
D-glucose do not reflect true absorption rates for this species. We
suspect that pre-systemic catabolism and/or sequestering of marker in tissues
(i.e. rapid removal from the bloodstream via hepatic and intestinal
first-pass effects) (Muratoglu et al.,
1986) could be responsible for these observations. We tested these
hypotheses by repeating experiments in red wattlebirds with the
non-metabolisable glucose analogue 3-OMG and tracing the movement of
radiolabel into the various tissues and organs at specific time points after
oral gavage.
We retrospectively calculated the potential impact of first-pass effects on
D-glucose bioavailability
(Gibaldi and Perrier, 1982) in
red wattlebirds, as a frame of reference for our measurements using
non-metabolisable 3-OMG. These calculations indicated that first-pass effects
could have reduced calculated bioavailability by at least 23–25%.
Subsequent measurements with 3-OMG (90% bioavailability, not
significantly different from D-glucose AE*) confirmed
that the erroneous result for D-glucose was indeed due to rapid
absorption and subsequent catabolism and/or removal of the latter via
hepatic and intestinal first-pass effects. However, as for the experiments
with D-glucose (trial A), the apparent rate of L-glucose
absorption exceeded that of 3-OMG, yielding values >1 for the
L/D ratio at the initial sampling time points. Although
3-OMG cannot be catabolised, the apparent absorption rate of 3-OMG calculated
at the initial sampling time points may not directly reflect that of
D-glucose. There are several concerns associated with the use of
3-OMG as an analogue for D-glucose. Firstly, the molecular mass of
3-OMG is slightly higher than that of D-glucose, lowering its
diffusion coefficient in water, although this can easily be corrected
(Chang and Karasov, 2004). The
paracellular pathway discriminates according to molecule size (e.g.
Chediack et al., 2003), so the
greater molecular mass of 3-OMG may lead to apparent paracellular rates of
absorption lower than for D-glucose. This effect may be especially
pronounced in species in which paracellular absorption is relatively more
important, and may thus lead to L/D ratios >1.
Secondly, because of this structural difference, 3-OMG has a lower affinity
for glucose transporters compared with D-glucose
(Ikeda et al., 1989;
Kimmich, 1981). In four mammal
species (laboratory rat, rabbit, guinea pig and hamster) and chickens (the
only avian species studied), averaging across species where data are available
for both D-glucose and 3-OMG in the same study and thus avoiding a
myriad of confounding methodological differences, the affinity (defined here
as 1/Km, or reciprocal of the Michaelis constant) of the
3-OMG for carrier-mediated glucose transport systems is 25% that of the
D-glucose (Bihler,
1969; Fedorak et al.,
1991; Shehata et al.,
1981; Thomson et al.,
1982). Lower affinity for glucose transporters may result in lower
uptake rates of 3-OMG relative to D-glucose. Furthermore, because
3-OMG apparently does not stimulate the recruitment of the GLUT2 transporter
to the apical membrane (Cheeseman and
Harley, 1991), the mediated component may be underestimated in
studies using only 3-OMG. However, we think this explanation is unlikely in
the present study because birds were fed on a sucrose maintenance diet
(hydrolysed to D-glucose and fructose in the intestine) immediately
before and after gavage with 3-OMG. Finally, a third possible explanation for
delayed 3-OMG appearance in plasma at initial sampling time points in red
wattlebirds could be accumulation in intestinal tissues. Boyd and Parsons
(Boyd and Parsons, 1979;
Boyd and Parsons, 1978)
demonstrated that as long as normal vascular perfusion is maintained, there is
no significant accumulation of this probe in anuran intestinal tissues during
steady-state in vivo perfusions (i.e. rate of appearance in blood
equals apical uptake). However, this may not apply for single-dose in
vivo uptake studies of compounds rapidly absorbed via mediated
mechanisms where there is loading of the probe into intestinal tissue pools
before washout into systemic circulation. In spite of these concerns, the
apparent rate of 3-OMG absorption was significantly higher than that of
D-glucose in red wattlebirds, and bioavailability calculated by our
pharmacokinetic methodology was comparable to D-glucose
assimilation efficiency by mass balance. Thus, 3-OMG appears to provide the
best available estimate of in vivo glucose absorption rate of these
two molecules.
A significantly higher proportion of 14C (administered as
radiolabeled D-glucose) was recovered in the liver and intestinal
tissues of the red wattlebird than the rainbow lorikeet, lending additional
support to the hypothesis that first-pass effects partly account for the low
oral bioavailability and calculated apparent rate of D-glucose
absorption. Although we collected gut contents and all major organs, we
clearly did not succeed in locating the bulk of the radiolabel administered to
rainbow lorikeets a short time after oral administration (for example, we
recovered a total of only 0.6% of 14C 2.5 min after oral
administration of D-glucose, or 13% by 5 min;
Fig. 3F). By contrast, in the
red wattlebird we recovered a total of 39% of 14C only 2.5 min
after oral administration of D-glucose
(Fig. 3F), with 17% of
14C recovered from liver, kidney, muscle and brain tissues
(Fig. 3D,E). Therefore, in
rainbow lorikeets, absorption and utilisation of radiolabeled glucose appears
to be a relatively slow process compared with red wattlebirds. Furthermore,
whereas we recorded an increase in radiolabel from both L- and
D-glucose in body tissues over time for the rainbow lorikeet, the
proportion of radiolabel recovered from red wattlebirds declined over time
(Fig. 3F). These data suggest
that rapid catabolism of D-glucose and removal from the body as
14CO2 was occurring in red wattlebirds, but over the
same timeframe rainbow lorikeets were still absorbing glucose from the
alimentary canal. This probably reflects differences in the physiological
mechanisms of glucose uptake, but may also reflect differences in digesta
passage rates between the species (not measured in red wattlebirds, mean
retention time of 0.4 mol l–1 sugar nectar diet in rainbow
lorikeets is 0.9 h, increasing to 1.6 h on 1.2 mol l–1 sugar)
(Karasov and Cork, 1996).
Although the use of radiolabeled D-glucose appeared to be an
improved method to avoid concerns with the commonly used 3-OMG, rapid
absorption and catabolism of the former in the red wattlebird resulted in a
significant underestimation of bioavailability. The assumption that the rate
of labelled D-glucose appearance in plasma reflects apical uptake
in the intestine is therefore not valid in this species. We conclude that
radiolabeled D-glucose is not an appropriate probe for in
vivo uptake measurements in animals where glucose is absorbed and/or
catabolised extremely rapidly. Overall, there appears to be no real advantage
in using radiolabeled metabolisable D-glucose over 3-OMG. In the
American robin, the difference in the estimated contribution of paracellular
absorption did not differ significantly between the two probes
(McWhorter et al., 2008),
however there is clearly a risk of erroneous results in birds such as the red
wattlebird.
Simultaneous administration of D-glucose analogues with
L-glucose provides a robust tool to evaluate the nutritional
significance of paracellular absorption, avoiding concerns that non-mediated
probes may be absorbed more slowly or in a different region of the intestine
(Chang and Karasov, 2004;
Schwartz et al., 1995). Our
data revealed that glucose absorption in rainbow lorikeets and red wattlebirds
is an extremely dynamic process. In spite of similar body masses and a similar
nectar diet, differences in handling glucose were apparent between our study
species. There appeared to be a shift in the relative importance of
paracellular uptake over time in rainbow lorikeets, probably the result of
decreasing substrate concentrations and thus the relative saturation mediated
glucose transporters over the absorptive phase. In red wattlebirds, although
the L/D-glucose cumulative fractional absorption ratio
exceeded 1 in this study (and thus provided no useable data on the
proportional contribution of paracellular to total glucose uptake), the fact
that the apparent absorption rate of L-glucose exceeded that of
D-glucose and 3-OMG suggests very significant reliance of
paracellular uptake. The apparent rate of 3-OMG absorption in red wattlebirds
exceeded that of D-glucose in rainbow lorikeets, and similarly the
rate of L-glucose absorption in wattlebirds exceeded that in
lorikeets. These observations suggest that glucose absorption overall occurs
more rapidly in wattlebirds than in lorikeets. These relatively large
nectarivorous birds, with their simple diets and gut structure may be ideal
model species for studying the mechanisms and regulation of paracellular
nutrient absorption.
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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