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Department of Psychology, Brooklyn College and The Graduate School, City University of New York, Brooklyn, NY 11210
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • flavor conditioning • fructose • saccharin • metabolism • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Consequently, lactose consumption by
postweaning rats leads to malabsorption of the sugar, which can produce
intestinal discomfort. This is indicated by reports that postweaning
rats rapidly learned to avoid a high lactose diet in favor of a low
lactose diet even when the high lactose diet was sweetened with
saccharin (Blake and Henning 1985
). Also, adult rats
show a decreased acceptance for lactose solution after its initial
consumption (Pelchat et al. 1983
) and learned to avoid a
flavor associated with the intake of a high lactose diet
(DiBattista 1990
).
A recent study from our laboratory (Sclafani et al. 1999
) indicates that low lactase levels may not be the only
problem postweaning animals have in handling lactose.
Food-restricted adult rats were trained to drink differently
flavored saccharin solutions paired with intragastric
(IG)3
infusions of water and 160 g/L glucose, fructose and galactose
solutions. In subsequent choice tests (30 min/d), during which there
were no infusions, the rats strongly preferred the glucose-paired
flavor (by 91%) over the water-paired flavor, but showed no
preference for the fructose-paired flavor. This confirmed earlier
reports that glucose and fructose differ in their ability to condition
flavor preferences (Sclafani et al. 1993
). The
unexpected finding was that the same rats avoided the
galactose-paired flavor relative to the water-paired flavor;
their percentage of intake of the galactose-paired flavor was only
21%. They also preferred both the glucose- and fructose-paired
flavors to the galactose-paired flavor. The flavor avoidance
conditioned by the galactose infusions suggests that galactose has
aversive postingestive consequences in adult rats. Thus, even if adult
rats could digest lactose, they might avoid high lactose diets because
of the aversive effects of galactose.
Flavor avoidance produced by IG nutrient infusions must be interpreted
with caution because nutrients delivered by this route may not be
processed in a normal manner by the gastrointestinal tract
(Pelchat et al. 1983
, Ramirez 1985
). It
seemed unlikely, however, that the observed galactose-conditioned
aversion was the result of the infusion procedure per se because
glucose infusions in the same rats conditioned a flavor preference.
Another factor that may have contributed to the galactose-induced
flavor aversion was the concentration of the sugar infusions. Rats
normally obtain galactose in the form of lactose in milk. Rat milk
contains ~40 g/L lactose, which yields 20 g/L galactose
(Newburg and Neubauer 1995
). Thus, the rats may have had
difficulty processing the much higher galactose concentration (160 g/L)
used in the infusion study. In addition, lactose digestion yields both
glucose and galactose, and the presence of glucose may affect the
postingestive actions of galactose. These issues were addressed in this
study by determining the flavor conditioning effects of orally consumed
galactose using lower sugar concentrations and using a galactose +
glucose mixture. The conditioning effects of galactose were compared
with those of isocaloric fructose solutions and noncaloric saccharin
solutions. Fructose was used as the comparison sugar because it has
minimal postingestive reinforcing effects (Sclafani et al. 1993 and 1999
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female Sprague-Dawley rats born in the laboratory from stock obtained from Charles River Laboratories (Wilmington, MA) were used. (All experiments were approved by the Brooklyn College Animal Care and Use Committee.) The rats were housed individually in stainless steel cages kept in a room maintained at 21°C under a 12-h light:dark cycle with lights on at 0800 h. Fluid was available from one or two stainless steel spouts at the front of the cage, and powdered nonpurified diet (Laboratory Rodent Diet 5001; PMI Nutrition International, Brentwood, MO) was available in a glass jar positioned in the back of the cage.
Experiment 1. Preference conditioning with 80 g/L sugar solutions.
The basic conditioning procedure involved giving the rats one-bottle training sessions with differently flavored galactose, fructose and saccharin solutions, which allowed them to associate the flavor of the solutions with their postingestive consequences. Flavor preferences were assessed in a series of two-bottle choice tests with the various solutions. Galactose is less sweet than fructose; therefore, saccharin was added to the galactose solution to improve its palatability. The test solutions contained 80 g/L galactose + 2 g/L saccharin (G), 80 g/L fructose (F) and 20 g/L sodium saccharin (S), all from Sigma Chemical (St. Louis, MO). (A pilot study determined that naive rats prefer the 80 g/L galactose + 2 g/L saccharin solution to the 80 g/L fructose solution, and prefer both to the 2 g/L saccharin solution.) The test solutions were flavored with 0.5 g/L cherry, grape or orange Kool Aid mix (General Foods, White Plains, NY). The solutions were prepared using tap water. The flavors or conditioned stimuli (CS) added to the galactose and fructose are referred to as the CS+G and CS+F, respectively, and the flavor plus sugar solutions are referred to as CS+G/G and CS+F/F, respectively. CS- refers to the flavor added to the nonnutritive saccharin solution, and CS-/S refers to the flavor + saccharin solution. The specific flavor (orange, grape or cherry) mixed into each solution was counterbalanced across subjects.
The rats (n = 8, 80 d old) were maintained at 85–90% of their free-feeding body weights by giving them restricted food each day. Water was freely available except during the daily 30-min test sessions. The rats were adapted to drink an unflavored 2 g/L saccharin solution during daily 30-min sessions. Their pretraining preference for the flavored galactose (CS+G/G) and flavored fructose (CS+F/F) solutions was determined in a two-bottle choice test. This and subsequent choice tests were conducted in two 30-min daily sessions with the left-right position of the solutions counterbalanced over sessions. A one-bottle training period of 12 d followed, during which the rats were fed 20 mL of CS+G/G, CS-/S, and CS+F/F on successive days in four 3-d cycles. For half the rats, the CS+G/G was given on d 1, CS-/S on d 2, and CS+F/F on d 3 of each cycle; the order of presentation was reversed for the remaining rats. Solution intakes were recorded after 30 min, and the bottles remained available overnight along with water and the daily food ration. The rats finished the solutions overnight without exception.
After training, the rats were given another two-bottle choice test with the CS+G/G and CS+F/F. This was followed by a choice test with the CS+G and CS+F flavors, both presented in 2 g/L saccharin solutions; these solutions are referred to as CS+G/S and CS+F/S. The rats were then given six additional one-bottle training sessions with the CS+G/G, CS+F/F and CS-/S solutions, presented as during the original training except that the solutions were available for only 30 min/d and intakes were unlimited. Next, half of the rats were given a two-bottle choice test with the CS+G/S vs. CS-/S, followed by a choice test with the CS+F/S vs. CS-/S. The remaining rats were given the tests in the opposite order.
Experiment 2. Preference conditioning with 20 g/L sugar solutions.
New rats (n = 9; 120 d old) were trained as in Experiment 1 except that 20 g/L galactose and 20 g/L fructose solutions were used. The 20 g/L galactose (G) solution also contained 2 g/L saccharin, and the fructose (F) solution contained 0.5 g/L saccharin. (Pilot work indicated that naive rats prefer 20 g/L galactose + 2 g/L saccharin to 20 g/L fructose + 0.5 g/L saccharin.) In addition, the rats were given access to 80 mL/d of the CS+G/G, CS+F/F and CS-/S solutions during the initial 12-d training period. Thus, the total amount of sugar (1.6 g/d) available to the rats was identical to that provided in Experiment 1.
Experiment 3. Preference conditioning with 20 g/L sugar solutions mixed with 20 g/L glucose (g).
New rats (n = 9; 120 d old) were trained as in Experiment 2 except that the sugar solutions contained 20 g/L galactose, 20 g/L glucose, and 2 g/L saccharin (Gg) or 20 g/L fructose, 20 g/L glucose, and 0.5 g/L saccharin (Fg). Also, the final CS+ vs. CS- preference tests were not conducted because the rats drank relatively little of the CS-/S solution during the initial 30-min training sessions.
Data analysis.
Two-bottle intake data were averaged over the two sessions for each test. The data from the pre- and posttraining flavored sugars tests and CS+ vs. CS- tests were analyzed by separate repeated-measures ANOVA followed by simple main effects tests, where appropriate. Intakes of the CS+ solutions in the two-bottle tests were also expressed as a percentage of total intakes. The data from the CS+G/S vs. CS+F/S tests were evaluated by Student's t tests. Solution intakes during the one-bottle trials were averaged over all of the sessions with each solution and analyzed by repeated-measures ANOVA. In addition, sugar intake was expressed in g/kg body weight. The criterion for significance in all tests was P < 0.05. Values presented in the text are means ± SEM
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The rats' preference for the flavored 80 g/L sugar solutions changed
reliably from before to after one-bottle training (P
< 0.01, Fig. 1
). In the pretraining test, they drank slightly more (P
= 0.08) CS+G/G than CS+F/F, but in the posttraining test, they
consumed more (P < 0.05) CS+F/F than CS+G/G. Their
percentage of CS+G/G intake declined from 76% (pretraining) to 18%
(post-training). When given the choice of the galactose- and
fructose-paired flavors presented in the saccharin solution, the
rats consumed more CS+F/S than CS+G/S (P < 0.01, Fig. 1
).
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Galactose and fructose intakes during the initial 30-min access periods on the one-bottle training days did not differ at 3.7 ± 0.5 and 3.9 ± 0.6 g/kg body weight, respectively. The rats consumed all of the allotted solutions (20 mL/d) during training; their 24-h sugar intakes were 6.9 ± 0.02 g/kg. During the 30-min retraining sessions, the rats consumed less galactose than fructose (3.1 ± 0.6 vs. 5.1 ± 1.0 g/kg; P < 0.001).
Preferences for the CS+ vs. CS- flavors, all presented in saccharin,
differed as a function of CS+ type (P < 0.01, Fig. 1
).
That is, the rats consumed more (P < 0.05) CS+F/S than
CS-/S, but more (P < 0.05) CS-/S than CS+G/S.
Compared with the CS-, the percentage of intake of the CS+F flavor was
69%, and of CS+G flavor was 29%.
Experiment 2. Preference conditioning with 20 g/L sugar solutions.
The rats' preference for the flavored 20 g/L sugar solutions changed
reliably from before to after one-bottle training (P
< 0.005, Fig. 2
). They drank more (P < 0.05) CS+G/G than CS+F/F before
training, but more (P < 0.05) CS+F/F than CS+G/G after
training. The percentage of CS+G/G intakes declined from 74%
(pretraining) to 18% (post-training). The rats also tended
(P = 0.12) to drink more CS+F/S than CS+G/S (Fig. 2)
.
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Galactose and fructose intakes during the initial 30-min access periods on one-bottle training days did not differ at 0.8 ± 0.1 and 0.7 ± 0.1 g/kg body weight, respectively. The rats consumed nearly all of the allotted solutions (80 mL/d) during training and their 24-h intakes of galactose and fructose did not differ at 5.4 ± 0.2 and 5.8 ± 0.1 g/kg, respectively. However, during the 30-min retraining periods, the rats consumed less galactose than fructose (0.5 ± 0.1 vs. 1.3 ± 0.2 g/kg; P < 0.01).
Preferences for the CS+ vs. CS- flavors, all presented in saccharin,
differed as a function of CS+ type (P < 0.01, Fig. 2
).
That is, the rats consumed more (P < 0.05) CS- than
CS+G/S, but their intakes of CS+F/F and CS- did not differ. Compared
with the CS-/S, the percentage of intake of CS+G/S was 32% and of
CS+F/S was 55%.
Experiment 3. Preference conditioning with 20 g/L sugar solutions mixed with 20 g/L glucose.
The rats' preference for the flavored mixed sugar solutions changed
reliably from before to after one-bottle training (P
< 0.01). They drank more (P < 0.05) CS+G/Gg than
CS+F/Fg before training, but more (P < 0.05) CS+F/Fg
than CS+G/Gg after training. The percentage of CS+G/G intake declined
from 63% (pretraining) to 28% (posttraining). The rats did not differ
in their intake of CS+F/S and CS+G/S (Fig. 3
). Intake during the test with the flavored saccharin solutions was
substantially less than intake during the flavored mixed sugar +
saccharin tests.
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The rats tended to consume less galactose than fructose during the initial 30-min access periods on one-bottle training days (1.3 ± 0.1 vs. 1.5 ± 0.1 g/kg body weight, P = 0.06). Total 24-h intakes of the sugars were both 6.6 ± 0.1 g/kg. Glucose intakes during the 30-min and 24-h training periods were equal to the galactose and fructose intakes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
These data are consistent with our previous finding that rats learned
to avoid a flavored saccharin solution that was paired with concurrent
IG infusions of galactose (Sclafani et al. 1999
). In the
earlier study, the rats were infused with a 160 g/L galactose solution
as they drank flavored saccharin (the CS+G). The IG infusion was
matched to the oral intake so that the net sugar concentration in the
stomach was 80 g/L, the concentration used in Experiment 1 of this
study. The results with the orally consumed sugar (Experiment 1) and
the IG infused sugar (Sclafani et al. 1999
) were
remarkably similar. That is, in Experiment 1, the rats showed a
post-training preference of 82% for the flavored fructose solution
over the flavored galactose solution; in the IG experiment, the
preference was 80% for the fructose-paired flavor over the
galactose-paired flavor. Also, the rats showed a 71% preference
for the CS- flavor over the galactose-paired flavor in Experiment
1, and a 79% CS- preference in the IG experiment. Furthermore, in the
one-bottle retraining sessions, the rats consumed more flavored
fructose than flavored glactose in Experiment 1 (18.7 vs. 11 mL/30 min)
and consumed more flavored saccharin + IG fructose than flavored
saccharin + IG galactose (17.9 vs. 14.4 mL/30 min) in the IG
experiment. The amounts of galactose consumed during the 30-min
training sessions of the two studies were also comparable (3.1 and 3.2
g/kg body weight).
Although the 80 g/L sugar concentration studied in Experiment 1 is at
the low end of the concentration range typically used in conditioning
studies with sugars, it is four time the galactose concentration in rat
milk (40 g/L lactose yields 20 g/L galactose) (Newburg and Neubauer 1995
). Experiment 2 revealed, however, that even at 20
g/L, galactose conditioned a flavor avoidance. Furthermore, the
magnitude of the galactose avoidance was similar to that produced by
the 80 g/L concentration in Experiment 1. The percentage of intake of
the CS+G/G, relative to the CS+F/F, was 18% in both experiments, and
the percentages of intake of CS+G/S relative to CS-/S were 29 and
32%. Although the rats were given the same amount of sugar (1.6 g/d)
in the two experiments, the rate of intake during training sessions was
lower with the 20 g/L solution than with the 80 g/L solution. In the
initial 30-min access periods on one-bottle training days, the rats
consumed 0.8 g/kg galactose in Experiment 2 compared with 3.7 g/kg in
Experiment 1. Thus, galactose avoidance is not influenced by
concentration over a range of 20–80 g/L, at least when total daily
dose is the same.
Rats normally consume galactose in the form of lactose, which yields both glucose and galactose when digested. Experiment 3 revealed that adding 20 g/L glucose to the 20 g/L galactose and fructose training solutions did not block the development of a galactose avoidance. The rats switched their preference from the flavored galactose + glucose to the flavored fructose + glucose solution after one-bottle training with the mixed solutions. The magnitude of the galactose avoidance was somewhat less with the mixed sugar solutions. That is, the posttraining percentage of intake of the flavored galactose solution, relative to the flavored fructose solution, was 28% in Experiment 3 compared with 18% in Experiment 2, but this difference was not reliable. Thus, at best, the presence of glucose only slightly attenuated galactose avoidance.
Fructose was used as a comparison sugar in this study on the basis of
earlier work showing that fructose has minimal postingestive
conditioning effects (Sclafani et al. 1993 and 1999
).
Although the rats in Experiment 1 developed a preference for the
fructose-paired (CS+F) over the saccharin-paired flavor (CS-),
other data indicate that this preference was due to the sweeter taste
of the 80 g/L fructose solution compared with the saccharin solution
(Sclafani and Ackroff 1994
). Note that in Experiment 2,
the rats did not prefer the CS+F flavor paired with the 20 g/L fructose
solution over the CS- flavor, despite the fact that the fructose
solution provided energy and the saccharin solution did not. Thus, the
switch in preference from the flavored galactose solution to the
flavored fructose solution is attributed to an aversive action of the
galactose rather than a positive postingestive action of the fructose.
This is further supported by the finding that not only did rats prefer
the CS+F flavor, they also preferred the saccharin-paired flavor
(the CS-) to the CS+G flavor in Experiments 1 and 2.
Although these results, together with our recent IG infusion data
(Sclafani et al. 1999
), indicate that galactose has an
aversive postingestive action in adult rats, the source of this action
is not certain. Galactose, like glucose, is readily absorbed in the
intestinal tract (Niewoehner et al. 1990
,
Niewoehner and Neil 1992
). Thus, malabsorption, which is
responsible for lactose intolerance, is not the cause of galactose
aversion. Galactose is metabolized primarily in the liver where it is
converted to glucose or stored as glycogen (Niewoehner and Neil 1992
). Like lactase, the enzymes required for galactose
metabolism (galactokinase, galactose-1-phosphate uridyltransferase and
uridine-5'-diphosphate galactose-4-epimerase) are most active during
the suckling period; at the end of that period, the levels decline
rapidly (Berman et al. 1978
). As a result, adult rats
metabolize galactose more slowly than glucose (Berman et al. 1978
, Niewoehner et al. 1990
, Niewoehner and Neil 1992
). Niewoehner et al. (1990)
reported that rats given an oral load of galactose (4 g/kg) showed high
circulating galactose concentrations that resulted in galactosuria. In
Experiment 1, the rats consumed nearly this amount of galactose (3.7
g/kg) during the initial 30-min access periods on one-bottle
training days. It may be, therefore, that the slow and incomplete
metabolism of galactose generated aversive unconditioned stimuli that
were responsible for the learned flavor avoidance. In rare hereditary
disorders, human infants lack the enzymes needed for galactose
metabolism and galactose ingestion results in galactosemia, a toxicity
syndrome that includes vomiting, liver disease and inanition among its
symptoms (Segal and Berry 1995
). Adult rats may
experience a mild form of galactosemia, which is revealed by the flavor
conditioning paradigm.
In summary, orally consumed galactose as well as intragastric galactose
infusions (Sclafani et al. 1999
) appear to have aversive
postingestive consequences in adult rats as revealed by conditioned
flavor preference tests. This can account for earlier reports that rats
consumed substantially less of the galactose solutions than of the
glucose solutions even when saccharin was added to the galactose to
improve its palatability (Debnam and Levin 1976
,
Richter and Campbell 1940
). The slow and incomplete
metabolism of galactose in adult rats is presumably responsible for the
sugar's aversive effects but this requires further investigation.
Lactose intolerance in animals and humans has been the subject of
considerable research (Rozin and Pelchat 1988
,
Saavedra and Perman 1989
). The present data indicate
that adult rats also experience aversive consequences after consuming
galactose, which would contribute to their avoidance of high lactose
diets. Whether galactose produces aversive effects in other species
remains to be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CS, conditioned stimulus;
CS+, flavor paired with sugar; CS- flavor paired with saccharin; F,
fructose; g, glucose; G, galactose; IG, intragastric; S, saccharin.
Manuscript received February 16, 1999. Initial review completed April 12, 1999. Revision accepted May 11, 1999.
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REFERENCES |
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