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RESEARCH REPORT |
1 Institute of Dentistry, PO Box 5281, FIN-90014 University of Oulu, Finland;
2 Oulu University Hospital, Oulu, Finland;
3 Laboratory, Oulu University Hospital;
4 Oulu Municipal Health Centre, Oulu, Finland; and
5 Department of Endodontics, Faculty of Dentistry, University of Toronto, ON, Canada;
* corresponding author, liisa.valikangas{at}oulu.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: dentin formation • sucrose • insulin • mineral excretion • rat
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanism behind the effect of a high-sucrose diet on dentin formation is not known (for review, see Tjäderhane et al., 2000). Previous studies have shown that caries lesions did not cause a reduction in dentin formation (Tjäderhane et al., 1994; Pekkala et al., 2000a; Huumonen et al., 2001). Instead, sucrose might have a direct effect on odontoblast metabolism, since glucose decreases type I collage synthesis in mature human odontoblasts in vitro (Välikangas et al., 2001). On the other hand, a high-sucrose diet causes hyperinsulinemia and insulin resistance in rats, leading to glucose intolerance and hyperglycemia (Gutma et al., 1987; Grimditch et al., 1988; Barnard et al., 1993). Furthermore, an increased blood glucose level induces calcium excretion to urine (Holl and Allen, 1987; Pekkala et al., 2000b). Hyperinsulinemia, in turn, inhibits renal tubular re-absorption of calcium (Leman et al., 1970; De Fronzo et al., 1975). The diminished levels of calcium available for mineralization may be a reason for the adverse effect of sucrose on the mineralized tissues, since bone and dentin have several biological similarities in health and disease (Larmas, 2001). Even though the direct effect of insulin on odontoblast metabolism has not been studied in vivo, the in vitro studies indicate that insulin per se does not affect odontoblast collagen synthesis (Välikangas et al., 2001).
While both a high-sucrose diet and hyperinsulinemia may have significant effects on bone metabolism, and a high-sucrose diet is known to affect dentin formation, the effect of hyperinsulinemia on dentinogenesis and/or dentinal caries progression is not known. The aim of this study was to evaluate the separate and combined effects of sucrose and insulin on dentin formation and mineral metabolism in growing rats. Based on the data available, the hypothesis was set that high dietary sucrose would affect dentin formation independently of insulin. To test the hypothesis, we studied the effect of exogenous insulin with and without high dietary sucrose load in growing rats.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The detailed descriptions of the diets have been published previously (Pekkala et al., 2000b). The first group was fed a standard rodent diet (Lactamin R 36, Ewos, Stockholm, Sweden), containing 34% barley flour, 43% wheat flour, 5.0% wheat grains, 4.5% vitamins and trace elements, 5.0% soya, 4.0% fish powder, and 4.5% other ingredients (further referred to as the control group). The second group was fed the same diet but also received daily subcutaneous insulin injections (1 U/100 g, 40 U/mL; Caninsulin Intervet Int. B.W., Boxmeer, The Netherlands) (further referred to as the insulinemic control group). The third group received a diet containing 41% sucrose (further referred to as the sucrose group), 10.1% barley flour, 10.3% wheat flour, 5.0% wheat grains, 5.0% wheat brans, 5.0% wheat fodder meal, 5.0% soya, 4.0% fish powder, 4.3% vitamins and trace elements, 4.5% other ingredients, and 5.8% casein, which was added to compensate for the loss of protein caused by the reduction of wheat and barley flour from the standard diet (Ewos R 642, Ewos, Stockholm, Sweden). The fourth group received the same high-sucrose diet, but was also given the daily subcutaneous insulin injections as above (further referred to as the insulinemic sucrose group). The groups not injected with insulin received respective subcutaneous injections of physiological saline. All diets were in powder form, and food and tap water were available ad libitum. The animals were housed 2 or 3 per cage under normal atmospheric conditions at 21°C and subjected to the same light/dark cycle (12 hrs light/12 hrs dark) and the same times of feedings, handling, and noise level.
At the ages of 28, 31, 35, and 38 days, animals were individually housed in metabolic cages for a 12-hour nocturnal period, and the urine excreted during that time was measured and stored at -20°C for further analysis.
Sample Collection and Preparation
Four wks after being weaned, the animals were anesthetized with a mixture of midazolam (Dormicum®, Roche, Basel, Switzerland) and Fentanyl-fluanizone (Hypnorm®, Janse Pharmaceutica, Brussels, Belgium) and sterile water (1:1.2 at 0.3 mL/100 g, i.p.). Blood was drawn from each pup by cardiac puncture, and the animals were then decapitated. After 10 min, the blood was centrifuged and the serum glucose (mmol/L) was measured by glucometer (Super GlucocardTM II, Arkray Factory Inc., Kyoto, Japan). The sera were stored by being frozen for further analysis. The jaws were de-fleshed and preserved in absolute ethanol.
Sample Measurements
Serum and urine insulin levels (ng/mL) were determined by means of a radioimmunoassay (Rat Insulin RIA Kit, Lincoln Research Inc., St. Charles, MO, USA), which utilizes an antibody made specifically against rat insulin. Urine glucose (mmol/L) was measured by means of an enzymatic method with glucose dehydrogenase (Hitachin 911, Boehringer Mannheim, Mannheim, Germany). Serum and urine phosphorus (mmol/L) were determined by a direct phosphomolybdate method (Roche Diagnostics, Rotkreuz, Switzerland) modified for a Cobas Integra 700 analyzer (F. Hoffmann-La Roche Ltd, Diagnostic Division, Basel, Switzerland). Serum and urine calcium (mmol/L) were determined by means of the o-cresolphthalein complexone (Roche Diagnostics, Rotkreuz, Switzerland) modified for a Cobas Integra 700 analyzer. Serum and urine potassium and sodium (mmol/L) were determined by ion-selective electrodes, with automatically diluted specimens modified for Cobas Integra 700.
To measure the amount of dentin formation, we sectioned the mandibles sagittally and measured the dentin formation under the main central transverse fissures of the first and second molars in the right mandible planimetrically, using a microscope (Leica DM RB, Leica Microscopie und systemic GmbH, Wetzlar, Germany) equipped with a computer-connected video image analyzer (Leica Q 500 MC, Leica Cambridge Ltd, Cambridge, UK) and fluorescent light with which the tetracycline stripes surrounding the dentin formed during the experiment could be seen (final magnification on screen, 113x) (Larmas and Kortelainen, 1989; Pekkala et al., 2000b). The number of main central fissures with dentinal caries lesions was calculated for each group, and the areas of dentinal caries lesions, seen as a change of fluorescence (Hietala et al., 1993), were measured planimetrically as described above.
Statistical Analyses
The statistical analyses were done with SPSS for Windows Release 9.0 (SPSS Inc., Chicago, IL, USA). The differences in mean weights, weight gains, dentin formation and serum phosphorus, potassium, calcium, sodium, and insulin between the groups were determined by one-way ANOVA with Tukey’s honestly significant difference (HSD) test. We determined daily urine and urine mineral excretion rates by counting the area under the urine excretion and urinary mineral level curve (AUC) for each animal and mineral. The differences in mean urine values between the groups were determined by one-way ANOVA. Wd determined the differences in the number of dentinal caries lesions using chi-square with Fisher’s Exact Test. The differences in the areas of dentinal caries, serum glucose, and urine insulin levels were determined by non-parametric Kruskal-Wallis ANOVA with the Mann-Whitney U test, because the data did not meet the assumption of homogeneity of variances.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), but dietary sucrose reduced it. The area of dentin formation was significantly (p < 0.001) smaller in the first (Fig. 1A) and second molars (Fig. 1B) of sucrose-fed and sucrose-fed insulinemic animals when compared with the controls and insulinemic controls.
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). The serum insulin levels in the insulin groups were significantly higher than in the other groups (p < 0.05) (Table 1). Sucrose diet increased serum insulin levels, but the differences between the sucrose-fed and control groups were not statistically significant. Serum phosphorus level was significantly lower in the control insulinemic group compared with the control group (p < 0.05), and serum sodium was significantly lower in the sucrose group compared with the control groups (p < 0.05) (Table 1). Urine excretion was increased in the insulinemic animals when compared with the other groups, but the differences were not statistically significant (Table 2). Exogenous insulin significantly increased excretion of insulin to urine (Table 2). No other differences in the urine mineral excretion rates were noticed among the groups. No differences in the final weights (mean ± SD) at the end of the experiment (control, 201.8 g ± 19.6; insulinemic control, 200.9 ± 31.3 g; sucrose, 213.6 ± 34.9 g; insulinemic sucrose, 213.6 ± 34.9 g) or in the weight gains during the experimental period were noticed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The serum glucose level was extremely low in the insulin groups at the end of the experiment (1.8-2.1 mmol/L, with the normal level ranking betwee 5.5 and 8.0), but despite the fact that the rats in the insulin groups appeared healthy, and the weight gains and serum and urine mineral levels were normal. Rapidly absorbed carbohydrates produce rapid increases in blood glucose levels, with a subsequent insulin response, and as a consequence blood glucose decreases (Wolever and Miller, 1995). Also, in this study, the serum insulin level was slightly higher in the sucrose group when compared with the control group, which is in agreement with the statement above. The blood glucose levels differed significantly between the insulinemic and non-insulinemic rats in both diet groups, since exogenous insulin decreased blood glucose levels regardless of the diet. Since the serum glucose levels were measured only once, at the end of the experiment, it is possible that adaptation to the high dietary sucrose or exogenous insulin levels would have occurred. Therefore, single-time-point analysis does not necessarily represent the situation during the earlier phases of the study. The possibility remains that the rapid peak in blood glucose level after sucrose diet ingestion may be enough to cause longterm effects on odontoblast metabolism, even after reduction in blood glucose level. This speculation is supported, in addition to our previously mentioned in vitro study (Välikangas et al., 2001), by a study in which the high-sucrose diet significantly reduced the dentin formation rate in rat molars when compared with a much more slowly absorbed high-starch diet, despite similar energy levels in the diets (Tjäderhane et al., 1994). It has been suggested that after a glucose load, glucose is rapidly transferred from blood into interstitial tissue, whence it may later be taken up by cells (Aussedat et al., 2000). This mechanism might explain the independent effects of a high-sucrose diet on dentin formation from the blood glucose levels. Further studies, with frequent control of blood glucose levels and other physiological parameters will be needed before final conclusions in this matter can be drawn.
In conclusion, exogenous insulin or hypoglycemia as such did not affect dentin formation. This study supports the hypothesis that a high-sucrose diet exerts a direct reducing effect on dentin organic matrix formation, and that exogenous insulin does not alter the effect of sucrose. Even though this study did not indicate that the detrimental effect of a high-sucrose diet on dentin formation could be related to the alterations in mineral metabolism, further research on this subject must be conducted before the final conclusions can be drawn.
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ACKNOWLEDGMENTS |
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Received October 10, 2001; Last revision May 9, 2002; Accepted May 23, 2002
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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| Journal of Dental Research ® | Critical Reviews (1990-2004) |
). The box reveals the first and third quartiles with median inside, and the "whiskers" show the highest and lowest values. Statistical analyses were performed by the Kruskal-Wallis ANOVA with the Mann-Whitney U test. N=7 in sucrose and 8 in other groups.