Chem. Senses 24: 387-392,
1999
© Oxford University Press 1999
Induction of Salivary Gurmarin-binding Proteins in Rats fed Gymnema-containing Diets
1 Department of Oral Physiology, School of Dentistry, Asahi University, Hozumi, Gifu 501-0296 2 Department of Physiology, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
Correspondence to be sent to: Dr Hideo Katsukawa, Department of Oral Physiology, School of Dentistry, Asahi University, Hozumi, Gifu 501-0296, Japan. e-mail:kat{at}dent.ashai-u.ac.jp
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Abstract |
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Gymnema sylvestre, a tropical plant, contains gurmarin that selectively suppresses sucrose responses of the chorda tympani nerve in rats and mice. We investigated preference for taste solutions and saliva composition in rats fed a diet containing this plant (gymnema diet). Preference for 0.01 M sucrose and a mixture of 0.03 M sucrose and 0.03 mM quinine-HCl significantly decreased at 1–2 days after the start of the gymnema diet and subsequently returned closely to the control levels within about a week. There was no significant change in preference for NaCl, monosodium glutamate and quinine-HCl during feeding trials. Submandibular saliva of rats fed the gymnema diet for 4 and 14 days showed an inhibitory effect on immunoreaction between gurmarin and antigurmarin serum. Analyses using electrophoresis and affinity chromatography indicated that the saliva contains gurmarin binding proteins with molecular weights of 15, 16, 45, 60 and 66 kDa. These results suggest that reduction of preference for sucrose was probably caused by gurmarin contained in the gymnema diet and subsequent restoration of the preference may be due to suppression of the effect of gurmarin by salivary gurmarin binding proteins induced by the gymnema diet.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Leaves of a tropical plant, Gymnema sylvestre, contain two types of specific inhibitors of sweet taste, gymnemic acid and gurmarin. Gymnemic acid, a mixture of triterpene glycosides, is a most potent inhibitor of sweet taste in humans and responses of the chorda tympani nerve to sucrose in chimpanzees (Hellekant et al.,1985
;
Kurihara, 1992). Gurmarin, a peptide of mol. wt 4209, is reported to
suppress selectively chorda tympani responses to sweeteners in rats (Imotoet al.,1991; Miyasaka and Imoto, 1995) and certain strains of
mice (Ninomiya and Imoto, 1995; Ninomiyaet al.,
1997, 1998). This inhibitory effect of sweetener responses
occurs at a low concentration (>1 x 10-6M) and lasts for >3 h
in the rat (Imotoet al.,1991). These sweet-taste inhibitors may
function as a defense against vermin damage by making the plant unpalatable to many species of
animals.
Our previous study (Ninomiyaet al.,1994) demonstrated
that when fed diets containing papain ( cysteine protease), rats did not soon consume the required
amounts of diet to maintain or increase body wt. However, some animals started to ingest
sufficient amounts of such diets at a few days after the start of feeding. At this time, cystatin S (a
cysteine protease inhibitor) was concomitantly induced in the submandibular saliva of animals.
It has been shown that cystatin S binds to papain and reduces its activity (Bobek and
Levine, 1992). We therefore proposed that cystatin S may participate in the reduction
of nociceptive stimulation of the oral mucosa by papain and improve food intake.
In this report, we investigate changes in taste preference and salivary composition in rats fed diets containing powder of leaves of G. sylvestre (gymnema diet). The results demonstrate that preference for sucrose transiently decreased and subsequently recovered at several days after the start of feeding at the time when gurmarin-binding proteins were induced in saliva. This suggests that these diet-induced salivary proteins influence behavioral preference for sucrose in rats.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Animals
Male Wistar rats (320–350 g body wt) were individually housed in plastic cages in a room maintained at 22–25°C with ~50% relative humidity. The room was lighted from 6.00 a.m. to 6.00 p.m. Animals were fed a commercial brand of non-purified diet ( CE-2, Clea Japan) for 2 weeks prior to experiments. In feeding trials (14 days), animals were divided into two groups, one of which was fed the commercial diet (control diet group) and the other diet supplemented with 3% ground (10 mesh) Gymnema leaves ( 3% gymnema diet group). In one experiment, animals fed diets containing 1 and 10% gymnema ( 1 and 10% gymnema diet groups) were also used. Table 1 gives the composition of the control diet.
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Preference test for a sucrose–quinine mixture
Preference behavior was routinely measured with a 48 h, two-bottle preference test. Rats
were presented with a choice between distilled water and a test solution for 48 h (Ninomiyaet al.,1989). The taste solutions (in distilled water) used were: 0.01, 0.03 and 0.1
M sucrose;
0.03 M NaCl; 0.03 M MSG; 0.003 mM quinine-HCl; and a mixture of 0.03 or 0.1 M sucrose and
0.03 mM quinine-HCl. Total intakes of each solution over 48 h were measured and used to
calculate preference percentages according to the following formula:
Preference percentage = volume of testing taste solution (ml) x 100/ total volume of testing taste solution and water (ml).
Preference percentages were compared before and during a feeding trial.
Collection of submandibular saliva
After the preference test, rats were starved overnight and then the submandibular ducts were
cannulated intraorally with polyethylene tubes (SP-8, Natsume, Japan) under pentobarbital
anesthesia (i.p., 40–50 mg/kg). Saliva was collected into a tube
containing ice-cold 10
mM citrate buffer (pH 4.5) for 1 h by evoking secretion with an i.p. dose of 20 mg/kg body wt of DL-isoproterenol–HCl (Ninomiya et al.,1994). Saliva samples were
stored at -80°C until analysis. The salivary glands were removed and weighed after
saliva collection. This procedure was carried out in both groups of rats.
Inhibition of the gurmarin–antigurmarin mouse antiserum reaction by saliva
Saliva (0.25–1 µl) was added to a reaction mixture of
gurmarin (10 ng) and
antigurmarin mouse antiserum (15 µl), and incubated at room temperature for 60 min
according to the method of Imoto et al. (Imoto et al.,1992). The precipitates were treated with biotin-labeled antimouse IgG rabbit antiserum
for 60 min and then with horseradishperoxidase-labeled streptoavidin (Histofine, Nichirei, Japan)
for 30 min. The peroxidase-labeled products were incubated with
2,2'-azino-di-[3-ethylbenzthiazoline sulfonate (6)] (Boehringer Mannheim, Germany) as
a
substrate in the presence of 0.01% H 2O 2 at room temperature for 15
min. Amounts of the precipitates in reaction with gurmarin and antigurmarin antiserum were
expressed as peroxidase activity (OD 415nm).
Affinity chromatography of salivary gurmarin-binding protein
Gurmarin (2 mg) was coupled to N-hydroxysuccinideactivated sepharose (1 ml, Pharmacia, Sweden) according to the manufacturer's instruction manual, except that 0.1 M phosphate buffer containing 0.5 M NaCl (pH 5.0) was used as a coupling buffer. Saliva samples were incubated with the gurmarin-coupled matrix at room temperature for 30 min. The column was washed with 50 ml of 0.1 M phosphate buffer containing 0.5 M NaCl (pH 5.0) and then with 0.1 M Tris–HCl buffer containing 0.5 M NaCl (pH 7.0) until the optical density at 280 nm of the eluate became zero. Gurmarin-binding proteins were eluted with 0.1 M Tris– HCl buffer containing 0.5 M NaCl and 2% hydroxypropyl ß-cyclodextrin.
For an electrophoretic analysis, the eluate (10 µg) from an affinity column was applied to
15% SDS–polyacrylamide gels prepared by the method of Laemmli (1970) and run at 20
mA for 60–90 min. Protein bands were visualized by silver staining
procedures using a
commercial kit (Sil-Best Stain, Nakarai Co., Japan).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Food intakes and increment of body wt in animals fed the 1, 3 and 10% gymnema diets were compared in order to choose the diet to be used for taste preference tests (Figure 1). There was no difference in daily intakes of diet between control diet groups (17.6 ± 1.5 g/day) and gymnema diet groups (1%, 20.1 ± 1.3; 3%, 18.0 ± 1.6; 10%, 20.9 ± 1.2 g/day) (Figure 1A). The intakes of any diet group remained unchanged from the beginning to the end of a trial. Increments of body wt in the 1 and 3% groups (58.7 ± 1.3 and 56.0 ± 3.3 g) were comparable to that (60 ± 5.8 g) in the control group in the 14 day feeding trials, but marked decreases were found in the 10% group (30.1 ± 4.5 g) (Figure 1B). In all of the following experiments, the 3% gymnema diet, having little or no effect on the growth of animals, was used as the experimental diet.
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Figure 2 shows changes in preferences for various sucrose solutions in animals fed the gymnema diets. Preference percentages for 0.1 M sucrose did not significantly change after the start of the gymnema diet (Student's t-test, P > 0.05). Similarly, no change was found in preference percentages for 0.03 M sucrose and for a mixture of 0.1 M sucrose and 0.03 mM quinine-HCl, although preference percentages for the single sucrose solution and the mixture were somewhat low compared with those for 0.1 M sucrose (Student's ttest, P > 0.05). Preference percentages for 0.01 M sucrose and a mixture of 0.03 M sucrose and 0.03 mM quinine-HCl decreased by 13.2% (Student's t-test, P< 0.01) and 23.3% (Student's t-test, P < 0.05), respectively, from the control levels at 1–2 days after the start of the gymnema diet. Subsequently, preference for 0.01 M sucrose and a mixture of 0.03 M sucrose and 0.03 mM quinine-HCl returned closely to the control levels within a week. As shown in Figure 3, preference percentages for 0.03 M NaCl, 0.03 M MSG and 0.003 mM quinine were not affected by the gymnema diet (Student's t-test, P > 0.05). Animals showed aversion for quinine independently of diet regime.
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): 0.1 M sucrose; 0.03 M Suc (
): 0.03 M sucrose; 0.01 M Suc
(
): 0.01 M sucrose; 0.1 M Suc + 0.03 mM Q (
): a mixture of 0.1 M sucrose and 0.03
mM quinine-HCl; 0.03 mM Suc + 0.03 mM Q (•): a mixture of 0.03 M sucrose and 0.03
mM quinine-HCl. Results were analyzed using Student's t-test. Each symbol
represents the average of 8–12 animals.
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Previous studies have revealed that salivary glands increase in weight and a new protein appears in their secretions in rats fed tannin- and papain-containing diet (Mehansho et al., 1983
; Ninomiya et al., 1994). In the
present study, however, there was no difference in the relative weight of the submandibular gland
between animals fed the control and gymnema diets (Figure 4). No
difference was also found in
the flow rate of salivary secretion (not shown). Figure 5 shows inhibitory
effects of rat
submandibular saliva on immunoreaction between gurmarin and antigurmarin antiserum.
Reduction of peroxidase activity shows inhibitory effects of saliva. When saliva collected at 4
and 14 days after start of the gymnema diet was added to the reaction mixture,
activities of peroxidase binding to reaction products were low in the gymnema diet groups
compared with the control diet group ( t-test, P < 0.05), suggesting that the
gymnema diet
increases inhibitory effects of saliva on the immunoreaction, but it does not increase the weight
of the submandibular glands.
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Gurmarin-binding protein (300 kDa), which suppresses the immunoreaction, is included in rat saliva (Imoto et al., 1992
). In order to confirm the presence
of this salivary protein, saliva
collected from rats fed the gymnema diet was eluted from a gurmarin-coupled Sepharose column
and subjected to electrophoretic analysis (Figure 6). Proteins tightly
absorbed on the resin were
effectively eluted from the affinity chromatography column with buffer with 2% hydroxypropyl
ß-cyclodextrin added, but not with the same buffer alone (data not shown). The eluate was
separated by SDS electrophoresis into five protein bands and their mol. wts were estimated at ,
15, 16, 45, 60 and 66 kDa. The 300 kDa, gurmarin-binding protein
was not found in the eluate. These findings suggest that there are at least five proteins with
affinity for gurmarin (putative gurmarinbinding proteins) in saliva of rat fed gymnema diet and
that they are different from the gurmarin-binding protein normally found in rat saliva.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Preference percentages for a lower concentration of sucrose (0.01 M) transiently decreased immediately after the start of the gymnema diet, although those for higher concentrations of sucrose (0.03 and 0.1 M) and other taste solutions were not significantly affected by the gymnema diet (Figures 2 and 3). Gymnema sylvestre leaves contain a water-soluble peptide (gurmarin) which selectively suppresses responses of the chorda tympani nerve to sweet taste stimuli in rats (Imoto et al., 1991
; Miyasaka and
Imoto, 1995) and certain strains of mice (Ninomiya and Imoto, 1995;
Ninomiya et al., 1997, 1998). Even
when the
tongue is continuously rinsed with water, the suppressive effects of this peptide last for several
hours. Gurmarin has been suggested to inhibit interaction between sweeteners and their receptor
proteins on the taste-cell membrane, because sucrose response suppressed by gurmarin is
recovered by rinsing the tongue with antigurmarin serum (Miyasaka and Imoto, 1995) and with ß-cyclodextrin which could form inclusion complexes with
gurmarin and reduce its effect (Ninomiya et al., 1998). The
decreases in
preference for lower concentrations of sucrose in the present study, therefore, are probably due to
the action of gurmarin on the taste-cell membrane and its inhibitory effects on sweetener
responses. The fact that gurmarin did not affect preference for higher concentrations of sugar
(0.03 and 0.1 M) may be due to the amount of gurmarin in the diet being barely enough to
suppress neural responses to sucrose at lower concentrations. Gurmarin content in dry G.
sylvestre leaves is about 10 p.p.m., which is estimated to be about 0.1 µM when the
leaves
are used in the 3% gymnema diet. On the other hand, in an electrophysiological study, gurmarin
could suppress sucrose responses of the chorda tympani nerve at >0.5 µM (Imoto et al., 1991).
In the present study, preference at 0.03 M sucrose was clearly decreased by the gymnema diet
when quinine was added, but not when the single solution was used (Figure 2). Suppression of
sucrose responses by gurmarin contained in the diet was probably not strong enough to override
peripheral neural responses that induce preference behavior to 0.03 and 0.1 M sucrose. However,
when quinine is added to the solution, the central nervous system would become susceptible to
reduction in preference behavior, because the neural responses to quinine are not affected by
gurmarin (Imoto et al., 1991) and the quinine solutions induce
avoidance
behavior (Figure 3).
The present study showed that decreased preference for sucrose returned to a control level within
a week or two after cessation of the gymnema diet (Figure 2) and that rat
saliva after the diet has
an inhibitory effect on immunoreaction between gurmarin and antigurmarin serum (Figure 5).
The time course of restoration of the preference was comparable with that of increases in such an
inhibitory effect of saliva, as shown in Figures 2 and 5. Inhibition of the immunoreaction by
saliva was suggested to be attributed to the gurmarin-binding protein (>300 kDa), which is
normally included in rat saliva at concentrations of >100 p.p.m. (Imoto et al., 1992). Also in the present study, electrophoresis showed five proteins with
affinity for gurmarin
(gurmarin-binding proteins, GBPs) in saliva of rats fed the gymnema diet. Their mol. wts
(15–66 kDa), however, were clearly different from that of the
gurmarin-binding protein
normally included in rat saliva, suggesting that the lower mol. wt proteins were newly induced by
the gymnema diet. Probably, GBPs decrease concentrations of free gurmarin in the diet and
suppress the activity of gurmarin, and, as a result, preference for sucrose returns to the control
levels. Reasons for the absence of the 300 kDa gurmarin-binding protein will be investigated in a
future study.
The parotid glands of rats maintained on tannin-containing diets show dramatic increases both in
weight and in amounts of proline-rich proteins (Mehansho et al., 1983).
Because the overall responses to tannins closely resemble the effects of isoproterenol (a
ß-adrenergic agonist) treatment, release of catecholamines induced by dietary tannins has
been
suggested to trigger production of proline-rich proteins in the salivary glands. Similarly,
enlargement of the submandibular gland and induction of cystatin S in rat saliva by dietary
papain was mimicked by isoproterenol treatment (Ninomiya et al., 1994). In the case of cystatin S, induction by dietary papain was suppressed by section of
the
glossopharyngeal nerve. Therefore, it has been suggested that chemosensory information for
papain is conveyed to the sympathetic center for salivation via the glossopharyngeal nerve and
then sympathetic impulses from there stimulate production of cystatin S in the submandibular
gland. On the other hand, weights of the submandibular glands remained unchanged after the
gymnema diet, although the PGBTs were induced. Therefore, the mechanism of induction of the
PGBTs could be different from those of proline-rich proteins and cystatin S.
Tannins contained in a diet induce proline-rich proteins in saliva of the rat (Mehansho
et al., 1983) and mouse (Mehansho et al., 1985), which bind
to tannins and suppress a variety of antinutritional effects [e.g. inhibition of activity of
alpha-amylase (Zhang and Kashket, 1989)]. Also, our previous study (Ninomiya et al., 1994) suggested that cystatin S is
induced in saliva of rats fed papain-containing diets. Cystatin S is known to suppress activities of
papain (a cysteine protease) contained in the diet. Thus, certain salivary proteins may be induced
in response to ingestion of aversive substances, such as tannin and papain, contained in food, and
resultant suppression of detrimental effects of the substances at oral level would serve as a
defense for the animals. Induction of the salivary GBPs may also represent a line of defense
against unfavorable reduction in the ability to discriminate nutrients such as sucrose. Purification
and further investigation of the salivary GBPs is in progress.
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Acknowledgments |
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We thank Dr Bruce P. Bryant for critical reading of the manuscript.
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References |
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Accepted March 30, 1999
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