| Sign In to gain access to subscriptions and/or personal tools. |
The Effect of a High-sucrose Diet on Dentin Formation and Dental Caries in Hyperinsulinemic Rats
1 Institute of Dentistry, PO Box 5281, FIN-90014 University of Oulu, Finland; Correspondence: * corresponding author, liisa.valikangas{at}oulu.fi
A high-sucrose diet decreases dentin formation and its minerals, but the mechanisms behind the effect are largely unknown. We studied the combined and separate effects of sucrose and insulin on dentin formation and mineral metabolism in growing rats. At weaning, animals were randomized into 4 groups: control/sucrose diets both with and without external insulin (1 U/x 100 g body weight daily). After 4 weeks, we measured areas of dentin formation, numbers and areas of dentinal caries lesions, and serum and urine glucose, insulin, Ca, Na, K, and P. Exogenous insulin increased serum and urine insulin levels and decreased serum glucose level, but did not affect dentin formation or dentinal caries lesion formation or progression. A high-sucrose diet decreased dentin formation independently of insulin. The differences in serum and urine minerals between the groups were minor. The findings confirm that sucrose-diet-induced reduction in dentinogenesis is independent of insulin and loss of minerals in urine.
Key Words: dentin formation • sucrose • insulin • mineral excretion • rat
A high-sucrose diet decreases dentin formation (for review, see Tjäderhane et al., 2000) and reduces its mineral elements and mineralization (Hietala and Larmas, 1995; Tjäderhane, 1996; Huumonen and Larmas, 1999) in young rats during primary dentinogenesis. The effect of sucrose is at least partly systemic (Pekkala et al., 2000a) and dose-dependent (Huumonen et al., 1997). A detrimental effect of dietary sucrose is common to the mineralized tissues, since a high-sucrose diet induces osteoporotic changes in bone, decreasing bone volume and thickness in hamsters (Saffar and Makris, 1982). Furthermore, it decreases Ca and P contents in rat bone and impairs the mechanical strength and mineralization rate in rat tibia and femur (Tjäderhane and Larmas, 1998). A high blood glucose level in diabetes is also known to disturb bone formation and its mineral density (Herrero, 1998; Verhaeghe et al., 2000). 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.
Animal Handling and Diets The Experimental Animal Committee of the Medical Faculty, University of Oulu, Oulu, Finland, approved the experimental procedures. Forty Sprague-Dawley rats (Mollegård Ltd, Ejby, Denmark) were weaned at the age of 21 days, weighed, marked, and randomized into 4 groups, 7 or 8 pups in each group. For the cariogenic challenge, all animals were inoculated with fresh suspension of Streptococcus sobrinus (ATCC 27531 K 1 Fitzgerald, American Type Culture Collection, Manassas, VA, USA) To mark the areas of dentin formation during the experiment, we gave each animal an intraperitoneal injection of oxytetracycline hydrochloride (30 mg/kg, Terramycin®, Pfizer, Brussels, Belgium) when weaned and 1 day before termination of the experiment. 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
Sample Measurements 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
Insulin alone did not affect the rate of dentin formation (Fig. 1  ), 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.
Sucrose feeding induced the occurrence and progression of dentinal caries. The occurrence of dentinal caries in the first and second molar central fissures was 6% (1/16) in the insulinemic control group, 50% (7/14) in the sucrose group, and 69% (11/16) in the insulinemic sucrose group. There were no dentinal caries lesions in the control group. The chi-square test indicated differences in the number of caries lesions between the diet groups, but with Fisher’s exact test, no statistically significant differences were observed between the insulinemic and non-insulinemic animals. The mean (± SD) areas (µm2 x 103) of dentinal caries lesions in the insulinemic sucrose group were slightly larger than in the sucrose group, but the difference was not statistically significant (Fig. 2 ).
The serum glucose levels in the insulin groups were significantly lower than in the sucrose and control groups (p < 0.01) (Table 1 ). 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.
Previous studies have demonstrated the reduction in dentin formation rate with high-sucrose diets (Hietala and Larmas, 1995; Pekkala et al., 2000a; Tjäderhane et al., 2000 [review]; Huumonen et al., 2001), but the mechanisms behind the effect have remained unknown. This study provides more detailed information about the mechanisms involved, demonstrating that insulin or hypoglycemia induced by insulin does not play a significant part in the regulation of dentin formation, since exogenous insulin did not affect dentin formation regardless of the diet. Furthermore, the effect of a high-sucrose diet does not seem to be mediated via its effect on mineral metabolism, since there were no differences in urinary minerals between the groups. Therefore, these in vivo results support the previous in vitro experiments indicating that reduced dentinogenesis may be caused by the direct effect of glucose on odontoblasts, reducing the synthesis of dentin organic matrix (Välikangas et al., 2001). 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.
This work was supported by the Academy of Finland and by the Finnish Dental Society. Received for publication October 10, 2001. Revision received May 9, 2002. Accepted for publication May 23, 2002.
Journal of Dental Research, Vol. 81, No. 8,
536-540 (2002)
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
). 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.