20 Research Report
Fluoride Vol. 32 No. 1 20-26 1999
EFFECTS OF SUBTOXIC DOSES OF FLUORIDE ON
SOME ENZYMES OF THE GLUCOSE METABOLISM
IN SUBMANDIBULAR SALIVARY GLANDS OF
FED AND OVERNIGHT-FASTED RATS
M V da Motta,* D N de Souza,* J Nicolau*
São Paulo, Brasil
SUMMARY: Effects of subtoxic doses of NaF on phosphofructokinase-1
(PFK-1), hexokinase (HK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) in the submandibular
salivary glands of fed and overnight-fasted rats were studied. After NaF injection, fed animals showed increased activity of PFK-1 in the glands at 1
hour and reduced activity was observed at 3 and 6 hours. In overnightfasted rats, PFK-1 exhibited increased activity in all groups. The activity of
HK increased in fed animals at 12 hours after one dose and at 24 hours after
two doses. One hour after NaF injection in fed animals, the activity of PK
increased. The activity of G6PD increased in the fed group 12 hours after
one injection and decreased in the fasted group after 3 and 6 hours. The activity of LDH increased only in the fed group 3 hours after NaF injection. We
conclude that the effect of fluoride on these enzymes in the submandibular
glands differs depending upon the nutritional state of the animals.
Key words: Fluoride injections, Fluoride in rats, Glucose metabolism, Rat saliva, Salivary
enzymes, Submandibular glands.
INTRODUCTION
An increase in dental fluorosis is occurring in developed countries due to
excessive ingestion of fluoride, mainly from formulations intended for topical
action.1-3 As with any pharmacological agent, the magnitude of metabolic effects of fluoride depends on various factors, such as the concentration in the
blood and the time necessary for excretion. The plasma concentration 30 min.
after ingestion reveals rapid absorption and generalized distribution. 4
At the cellular level, as an enzymatic inhibitor, there are conflicting reports
on the action of fluoride on adenyl cyclase. 5-7 Increased salivation in fluorideintoxicated individuals 4,8 as well as secretion of fluoride ion by salivary
glands,9 led some researchers to examine possible metabolic alterations in salivary glands, due to fluoride action.
Some reports point to a fluoride action on the salivary glands via adenyl
cyclase.10,11 However, by examining the effect of fluoride on glycogen metabolism in the submandibular glands, we verified that the action of this element
depends on the animal nutritional state, 12 and does not necessarily stimulate
this system.
The purpose of the present investigation was to extend our studies on the effect of subtoxic doses of fluoride on submandibular salivary glands by examining enzymes of the carbohydrate metabolism of fed and overnight-fasted rats.
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*Oral Biology Research Center, Faculty of Dentistry-USP,
Av. Prof. Lineu Prestes 2227 05508-900, São Paulo, Brasil.
Fluoride and salivary glucose metabolism
21
MATERIALS AND METHODS
Male Wistar rats weighing 175-220 g either fed ad libitum or denied food
overnight (15 hours) were injected with 0.2 mL NaF solution, equivalent to 5
or 10 mg F-/kg body weight (EXP, experimental) or isovolumetric amounts of
saline (C, control). After the animals were sacrificed by cranial traumatism,
the submandibular salivary glands were immediately removed, clamped between aluminum tongs, pre-cooled in dry ice, weighed avoiding thawing, and
homogenized at 10% (W/V) in a Potter type homogenizer with a teflon pestle.
The homogenizing medium contained 50 mmoles/L imidazole buffer pH 7.0,
1 mmol/L beta-mercaptoethanol and 2 mmoles/L EDTA. The homogenates
were centrifuged at 27,750 x g for 20 min. in a refrigerated centrifuge and the
supernatants used for enzymatic and protein determinations.
Enzyme Assays
Enzyme activities of various supernatants were assayed by changes in absorption at 340 nm due to reduction of NADP or oxidation of NADH. The
reaction was initiated by adding aliquots of homogenate supernatant to the
reaction mixture in the cuvette, and the absorbance was monitored for 10 min.
at 25oC in a Beckman DU 68 spectrophotometer, using a 1-mL cuvette with 1cm path length. One unit of enzyme activity to the amount of enzyme that
converts 1 mol of the substrate per min.; specific activity is expressed in
U/mg of protein. Enzyme activities (hexokinase, HK; phosphofructokinase-1,
PFK-1; pyruvate kinase, PK; lactate dehydrogenase, LDH, and glucose-6phosphate dehydrogenase, G6PD) were measured according to the methods
described elsewhere.13
RESULTS
The results obtained in this investigation are expressed in Tables 1 to 6.
When fluoride was injected into fed animals, observations included an increased activity of PFK-1 at 1 hour and a reduced activity in the glands of rats
sacrificed at 3 and 6 hours after injection (Tables 1 and 2). The activity of HK
increased at 12 hours after one dose and 24 hours after two doses of fluoride
(Tables 1 and 2). The activity of PK increased only 1 hour after one injection.
The activity of the other two enzymes, G6PD and LDH, increased at 12 hours
for G6PD and at 3 hours for LDH, after one injection of fluoride (Table 3).
With overnight-fasted rats a different pattern was observed. Only PFK-1
(Table 4) and G6PD (Table 5) showed variation. The activity of PFK-1 increased in all groups studied, and the activity of G6PD decreased in the
groups examined 3 and 6 hours after the injection of 10 mg F -/kg body
weight. Table 6 shows that blood glucose increased in the groups studied 1
and 3 hours after one injection of fluoride.
DISCUSSION
Reports reveal that for the liver, fluoride has an inhibitory effect on some
enzymes, such as PFK-1, PK, G6PD, enolase and phosphoglyceromutase.11,14-17
Under the same experimental conditions (excess vitamin A), we have shown
that the response of the submandibular gland is different from the liver. 18
Fluoride 32 (1) 1999
Groups
(Hours)
1
PFK-1 (mU/mg protein)
C
EXP
1H
(12) 13.11  1.34 (10) 13.26  1.70
3H
(11) 13.13  2.13
6H
(12) 13.75  1.78 (16) 14.63  1.57
12 H
2
HK (mU/mg protein)
C
EXP
24 H
(6) 16.63  0.71
PK (U/mg protein)
C
EXP
(7) 23.20  1.90* (13) 0.22  0.03
(8) 13.51  2.69 (11) 16.11  1.69 (19) 13.90  2.61*
(14) 0.27  0.03*
(9) 0.22  0.03
(15) 0.22  0.04
(8) 16.60  2.03 (17) 13.94  2.52* (15) 0.22  0.04
(10) 0.23  0.03
(9) 14.89  2.21 (11) 18.34  2.70* (12) 16.25  1.22 (15) 15.55  2.19 (10) 0.22  0.04
(11) 0.24  0.04
(12) 13.04  2.61 (11) 16.58  1.49*
(7) 15.88  1.98 (13) 15.91  2.95
(8) 0.22  0.03
(9) 0.21  0.03
Table 2. 10 mg F-/kg body weight
Groups
(Hours)
1
1H
(13) 13.11  1.34 (14) 13.72  2.99
3H
(10) 13.14  2.13
6H
(12) 13.74  1.78 (14) 13.72  2.11
12 H
2
HK (mU/mg protein)
C
EXP
24 H
(9) 14.89  2.21
PFK-1 (mU/mg protein)
C
EXP
(6) 16.63  0.71
(7) 21.79  3.17* (14) 0.22  0.03
(8) 12.79  0.99 (11) 16.11  1.69 (16) 13.54  2.92*
(13) 0.25  0.03*
(9) 0.22  0.03
(17) 0.23  0.03
(8) 16.60  2.03 (15) 13.95  2.94* (16) 0.22  0.04
(13) 0.22  0.04
(7) 18.77  1.67* (14) 16.25  1.22 (17) 17.59  2.18
(12) 13.06  2.61 (12) 17.23  1.36*
PK (U/mg protein)
C
EXP
(7) 15.88  2.95 (12) 15.91  2.95
(9) 0.22  0.03
(20) 0.23  0.02
(8) 0.22  0.03
(12) 0.21  0.02
da Motta, de Souza, Nicolau
Table 1. 5 mg F-/kg body weight
22
Fluoride 32 (1) 1999
Tables 1 and 2. Specific activity of hexokinase (HK), phosphofructokinase-1 (PFK-1) and pyruvate kinase (PK) in
submandibular glands of fed rats injected with a single dose and two doses of fluoride (5 or 10 mg F-/kg body weight)
(EXP) or NaCl solution (C), and sacrificed at the indicated times after injection. Values are mean  S.D. for the number
of samples indicated in parenthesis. The asterisks mean statistically significant by the Student’s “t” test (p < 0.01).
Table 3. Specific activity of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) in the
submandibular glands of fed rats injected with 5 mg F-/kg body weight (EXP5), 10 mg F-/kg body weight (EXP10) or
NaCl solution (C) and sacrificed at the indicated times following the injection.
Groups
(Hours)
1
2
C
G6PD (mU/mg protein)
EXP5
EXP10
C
LDH (U/mg protein)
EXP5
EXP10
(11) 17.95  2.38 (11) 17.45  3.76 (11) 18.71  2.84 (12) 0.27  0.02
(11) 0.26  0.03
3H
(9) 18.48  3.18 (10) 18.08  2.19 (10) 18.13  2.66 (10) 0.26  0.03
(11) 0.21  0.03*
6H
(12) 17.18  3.06 (15) 18.38  2.86 (15) 18.43  2.42 (16) 0.26  0.03
(17) 0.25  0.04
(17) 0.27  0.02
12 H
(12) 18.42  2.11 (17) 21.89  2.16* (20) 21.90  2.19*
(6) 0.26  0.02
(6) 0.26  0.04
(7) 0.27  0.02
24 H
(12) 18.87  2.84 (14) 20.61  1.44 (14) 20.08  1.72
(9) 0.27  0.02
(7) 0.25  0.03
(7) 0.26  0.03
1H
(8) 0.26  0.03
(8) 0.23  0.03*
Groups
(Hours)
HK (mU/mg protein)
C
EXP
(12) 20.08  1.85 (12) 19.74  2.36
3
(9) 21.68  1.82 (12) 22.25  1.75
6
12
PK (U/mg protein)
C
EXP
(9) 20.86  2.28 (11) 25.34  2.97*
(6) 0.29  0.02
(10) 0.29  0.03
(9) 20.23  2.07
(9) 26.17  3.00*
(7) 0.31  0.02
(10) 0.31  0.04
(10) 20.79  1.93 (11) 20.79  2.16
(9) 20.18  2.61 (12) 24.27  3.48*
(9) 0.30  0.03
(10) 0.33  0.04
(10) 21.23  1.19 (10) 22.03  2.56
(9) 22.54  2.69 (11) 28.91  3.19* (10) 0.30  0.03
(4) 0.29  0.02
Values for both tables are mean  S.D. for the number of samples indicated in parenthesis.
The asterisks mean statistically significant by the Student’s “t” test (p < 0.001).
23
Fluoride 32 (1) 1999
1
PFK-1 (mU/mg protein)
C
EXP
Fluoride and salivary glucose metabolism
Table 4. Specific activity of hexokinase (HK), phosphofructokinase (PFK-1) and pyruvate kinase (PK) in the submandibular glands of overnight fasted (15 hours) rats injected with 10 mg F-/kg body weight (EXP) or NaCl solution (C) and
sacrificed at the indicated times after injection.
24
da Motta, de Souza, Nicolau
Table 5. Specific activity of glucose-6-phosphate dehydrogenase (G6PD) and
lactate dehydrogenase (LDH) in the submandibular glands of overnight-fasted
(15 hours) rats injected with 10 mg F-/kg body weight (EXP) or NaCl solution (C)
and sacrificed at the indicated times after injection.
Groups
(Hours)
G6PD (mU/mg protein)
C
EXP
1
(8) 2.31  0.31
3
(9) 2.31  0.22
6
12
(10) 2.55  0.36
LDH (U/mg protein)
C
EXP
(7) 0.56  0.04
(8) 0.55  0.05
(8) 2.04  0.42*
(7) 0.55  0.04
(8) 0.55  0.07
(11) 2.39  0.29
(9) 2.17  0.45*
(7) 0.55  0.03
(9) 0.55  0.08
(10) 2.40  0.31
(9) 2.51  0.23
(6) 0.54  0.03
(10) 0.54  0.06
Table 6. Blood glucose concentration of overnight-fasted (15 hours)
rats injected with 10 mg F-/kg body weight (EXP) or NaCl solution (C)
and sacrificed at the indicated times after injection.
Groups
Glucose (mg/100 ml blood)
C
EXP
1
(5) 74.82  6.38
(6) 108.42  8.40*
3
(5) 72.75  4.89
(5) 101.38  11.51*
6
(4) 74.00  4.89
(4) 78.00  5.16
12
(4) 65.98  1.03
(4) 67.78  3.97
Values are mean  S.D. for the number of samples indicated in parenthesis.
The asterisks mean statistically significant by the Student’s “t” test (p < 0.01).
____________________________
In fluoride liver studies, other authors report a reduction of the glycogen
content.16,19,20 However, we found that administration of fluoride to rats increased the glycogen content of the submandibular gland, 12 and that the glycogenolysis depends upon the nutritional state of the animals.
Our results clearly show that the activities of both HK and PFK-1 in the
submandibular gland are influenced by fluoride. Animals injected with fluoride showed increased HK activity from one dose after 12 hours, and from 2
doses after 24 hours. HK is located in a crucial point for glucose utilization by
the cell, since it promotes the phosphorylation of glucose for subsequent use
in glycolysis. When fluoride was injected into overnight-fasted rats, no difference in the activity of this enzyme was found compared with the control.
On the other hand, the results obtained for PFK-1 were more evident. In
fed animals there was an increase in the activity of this enzyme in the groups
sacrificed after 1 hour and reduction in the groups sacrificed 3 and 6 hours
Fluoride 32 (1) 1999
Fluoride and salivary glucose metabolism
25
after fluoride injection. However, the administration of fluoride to overnightfasted rats showed increased activity of this enzyme in all groups sacrificed
after one injection. In overnight-fasted rats the injection of NaF causes a transitory hyperglycemia, thereby increasing the supply of glucose substrate for
the glands. This result led us to conclude that fluoride acts on the submandibular gland of fasting animals through different mechanisms from those observed in the activation of adenyl cyclase system.
Our data agree with the hypothesis21 that fluoride acts on the membrane
proton transport system through inhibition of ATPase/ATP synthesis. In this
condition, only a small amount of ATP would be formed at this level, requiring, therefore, a greater action by the glycolytic pathway.
The results obtained for G6PD showed an increased activity in the submandibular glands of fed animals sacrificed 12 hours after administration of
fluoride. As there was a reduction in PFK-1 activity in the groups sacrificed at
3 and 6 hours after injection of fluoride, we can conclude that G6P is being
diverted to the pentose phosphate pathway. On the other hand, in overnightfasted rats sacrificed at 3 and 6 hours following fluoride injection, there was a
reduction in the G6PD activity, indicating a prevalence of the glycolytic
pathway in this nutritional condition.
Concerning LDH, a bifunctional enzyme, a reduction in its activity is seen
only in fed animals, sacrificed 3 and 6 hours after fluoride injection.
In conclusion, under the experimental conditions of this study, we find that
fluoride acts differently on the enzyme activities of the submandibular glands,
depending upon the nutritional status of the animals.
ACKNOWLEDGEMENTS
This work was partially supported by CNPq. J. Nicolau holds a fellowship
from CNPq.
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Published by the International Society for Fluoride Research
Editorial Office: 17 Pioneer Crescent, Dunedin 9001, New Zealand
Fluoride 32 (1) 1999