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Glucose-induced inhibition of in vitro bone mineralization
Bone Vol. 28, No. 1 January 2001:21–28
Glucose-induced Inhibition of In Vitro Bone Mineralization E. BALINT,1,2 P. SZABO,2 C. F. MARSHALL,2 and S. M. SPRAGUE1,2 1
Department of Medicine and the 2Research Institute, Evanston-Northwestern Healthcare, Northwestern University Medical School, Evanston, IL, USA
Introduction Patients with diabetes tend to have an increased incidence of osteopenia that may be related to hyperglycemia. However, little is known about how glucose may alter bone formation and osteoblast maturation. To determine whether glucose affects osteoblastic calcium deposition, MC3T3-E1 cells were incubated in media containing either a normal (5.5 mmol/L) or high glucose concentration (15 mmol/L) or mannitol (15 mmol/L), and bone nodule formation was examined. Net calcium flux was measured thrice weekly and cumulative calcium uptake was determined. Compared with control incubations, glucose significantly inhibited daily and cumulative calcium uptake into the nodules. At the time of matrix maturation, cultures undergo a rapid phase of increased calcium deposition; this was significantly inhibited by the presence of glucose. Total calcium uptake, determined by acid digestion, was also significantly inhibited by glucose. Area and number of nodules were quantitated at the end of the incubation period (day 30) by staining with Alizarin Red S calcium stain. Compared with both control and mannitoltreated cultures, the number of nodules was increased by incubation with glucose. Furthermore, both the average total nodular area and calcified nodular area of large nodules were increased by glucose. Cellular proliferation as well as the release of markers of osteoblast activity (osteocalcin and alkaline phosphatase) were determined at the end of the experimental period (day 30). Cellular proliferation and alkaline phosphatase activity was significantly increased in the presence of glucose, however, the release of osteocalcin into culture media was similar in all three groups. In conclusion, the present study shows that elevated glucose concentration present throughout the development of murine osteoblasts stimulates cellular proliferation while inhibiting calcium uptake. The result of glucose inhibition of calcium uptake suggests that bone could be structurally altered in diabetes. (Bone 28:21–28; 2001) © 2001 by Elsevier Science Inc. All rights reserved.
Osteopenia has been associated with diabetes, however, data demonstrating a causal relationship are limited and controversial. Bone mineral density in patients with both type I and type II diabetes has been reported to be decreased by greater than 10% compared with gender- and age-matched healthy persons.17,18,20 Although most investigators agree that osteopenia occurs in patients with type I diabetes, those with type II diabetes may have normal or increased bone density.11,16,18,44,47 In those with type I diabetes, the degree of osteopenia correlates with duration of diabetes.13,52 Long-term bone loss has been reported to be more severe among patients with type I diabetes compared with those with type II.16,18 This discrepancy in bone mass between those with type I and type II diabetes may be due, in part, to the obesity frequently associated with type II diabetes, as obesity is associated with increased bone density,11,53 thus masking the osteopenic affect of diabetes. Epidemiological studies evaluating the relationship between diabetes and fractures are also conflicting. An increased prevalence of diabetes has been described in patients with osteoporotic fractures,3,15,21 however, diabetes does not appear to be an independent risk for osteoporotic fracture.9,12 The tendency that patients with diabetes may have increased fracture rates is demonstrated among patients undergoing solid organ transplantation. Fractures occur in 10 –30% of solid organ transplant recipients.10,29 However, in those with preexisting diabetes, the incidence of having at least one fracture is greater than 45% after renal transplantation.23,29 In patients undergoing kidney pancreas transplantation, up to 50% were reported to sustain at least one fracture within 5 years of transplantation.5,29 Although clinical data concerning the role of diabetes in causing bone disease are conflicting, it is likely that diabetes alters bone metabolism. Tamayo and colleagues found that osteoblastic collagen synthesis was disturbed in diabetes.42 Serum osteocalcin concentrations, a marker of osteoblastic activity, were significantly lower in spontaneously diabetic BB rats compared with nondiabetic rats.45 Smythe and colleagues reported a positive correlation between hyperinsulinemia and bone density; however, others were unable to confirm such a correlation.11,32,35 Hyperglycemia is also associated with hypercalciuria19,20,30 and impairment of vitamin D metabolism,20 factors associated with bone loss. Metabolic acidosis, which frequently accompanies hyperglycemia, is associated with bone loss in vivo25,33 and induces bone resorption while inhibiting bone formation in vitro.4,38 Very little is known about the effect of hyperglycemia on bone metabolism. Clinical studies concerning the correlation of bone mass and glycemic control are controversial. Wise et al. reported that linear growth velocity is dependent on metabolic
Key Words: Glucose; Osteoblast; Calcium; Bone nodule; Mineralization.
Address for correspondence and reprints: Dr. Stuart M. Sprague, Evanston Northwestern Healthcare, Division of Nephrology, 2650 Ridge Avenue, Evanston, IL 60201. E-mail: [email protected] *E. Balint’s current address: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA; P. Szabo’s current address: 2nd Department of Medicine, Semmelweis University Medical School, Budapest, Hungary. © 2001 by Elsevier Science Inc. All rights reserved.
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control in children with type I diabetes mellitus.51 Rosato and colleagues reported an increase of bone turnover after improving metabolic control of patients with type II diabetes.31 However, Okazaki et al. showed a decrease of bone turnover after metabolic improvement of poorly controlled type II diabetes mellitus.24 McNair et al. reported a correlation between loss of bone mass and fasting blood glucose concentrations.19 However, others were unable to demonstrate a correlation between glycosylated hemoglobin and change in bone mass.17,42 Morohoshi and colleagues demonstrated that high concentration of glucose induces the production of the potent bone resorbing cytokines, interleukin-6 (IL-6) and tumor necrosis factor-␣ (TNF-␣), from human monocytes.22 Glucose has been shown to have a direct activating effect on osteoclasts and seems to be the principal energy source for osteoclastic bone resorption.50 Even less information is available concerning the effect of glucose on bone formation. Thus, the purpose of the present study was to determine the effect of prolonged elevated glucose concentrations on osteoblastic bone formation. Using the in vitro bone nodule formation assay, these studies demonstrated that elevated glucose concentrations resulted in decreased calcium uptake with defective nodular mineralization. Materials and Methods Bone Nodule Formation Assay Bone nodule formation was induced as previously described.37,38 Briefly, MC3T3-E1 mouse osteoblast-like cells were plated at a density of 4.5 ⫻ 104 cells/35-mm Petri dish and incubated in ␣-MEM (containing 5.5 mmol/L glucose) supplemented with 10% fetal bovine serum, 50 IU/mL potassium penicillin, and 50 g/mL streptomycin (Life Technologies, Gaithersberg, MD). Cells were grown until confluent, designated as day 0, and nodule formation was induced by the addition of 50 g/mL ascorbic acid and 10 mmol/L -glycerophosphate to the medium. The dishes were randomly divided into three treatment groups: vehicle (control), 15 mmol/L D-glucose (glucose), or 15 mol/L D-mannitol (osmotic control). The progress of osteoblast development was monitored by measuring calcium deposition into the cultures (daily calcium uptake). Media was changed every 48 –72 h and the calcium content of the conditioned media determined. A culture dish incubated without cells and subjected to similar media changes was used for baseline calcium determinations (medium blank). The experiment was carried out until the stage of maximal calcium influx (day 30), and randomly selected culture dishes were stained using Alizarin Red S calcium stain. At the same time (day 30), another set of randomly selected culture dishes were subjected to acid digestion in order to assess the amount of total calcium content per culture dish. For osteocalcin and alkaline phosphatase determinations, conditioned media were collected and stored at ⫺70°C until analysis. Nodule Quantification At the end of the experimental period (day 30), randomly selected nodule culture dishes were stained for calcium with Alizarin Red S (Aldrich Chemical Company Inc., Milwaukee, WI) as previously described.6 Briefly, nodules were fixed in 10% buffered formalin, washed with distilled water, and stained with 2% Alizarin Red S for 2 min. Staining with Alizarin Red S is a standard method for visualization of nodular pattern and calcium deposition of osteoblast cultures in vitro.27 Alizarin Red S stains calcium dark red and the tissue yellow. The culture dish incubated without cells and subjected to similar media changes did not stain positive for calcium with Alizarin Red S (medium blank
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as negative control; data not shown). The number of calcifying foci per culture dish was counted macroscopically on a 5-mm grid. Culture dishes were also examined under 25⫻ and 50⫻ magnification to microscopically assess nodule number, calcified, and uncalcified area. The number and area of calcified foci was determined by video analysis using the Osteomeasure histomorphometric software (OsteoMetrics Inc., Decatur, GA) as previously described.48,49 Large nodules include calcifying foci larger than 0.1 mm2, and small nodules are calcifying areas smaller than 0.1 mm2. Total and calcified area of large nodules was determined from randomly selected culture dishes from two independent experiments (n⫽20). Number of small nodules was determined at 50⫻ magnification from 20 randomly selected visual fields from two independent experiments (n⫽20). Calcium Calcium concentrations were determined from conditioned media immediately after collection using automated fluorometric titration analysis (Calcette; Precision Systems, Sudbury, MA) as previously described.1,37,38 Daily calcium uptake of the cultures was calculated by subtracting the value of conditioned media for each culture dish from the value of medium blank. A positive flux (influx) indicates calcium uptake from the medium into the cultures. Cumulative calcium uptake values were calculated by summing the daily uptake values for each culture dish. Total calcium uptake was determined from randomly selected culture dishes at day 30 using automated fluorometric titration analysis after an overnight incubation with 2 mL of 2 N HCl.1,37,38 Proliferation Assay On day 30, DNA synthesis was measured by pulse labeling the cultures with 10 Ci of 3H-labeled thymidine (DuPont NEN, Boston, MA) for 2 h as previously described.37 At the end of the incubation period, medium was aspirated and cells washed in PBS, then incubated in 5% trichloroacetic acid (TCA) for 20 min. The cells were then passed through glass-fiber filters that had been presoaked with 5% TCA. Filters were then washed with 95% ethanol, dried overnight, and placed into 10 mL of liquid scintillation cocktail (EcoLume; ICN, Costa Mesa, CA). Radioactivity of the filters was determined by liquid scintillation spectrometry. Because the amount of extracellular matrix proteins interferes with total cellular protein determination, the data were determined as total 3H-labeled thymidine incorporation per culture dish and expressed as percentage of control. Osteocalcin Osteocalcin content of the medium was measured using a mousespecific IRMA (Immutopics, Inc., San Clemente, CA). Briefly, sample containing mouse osteocalcin was incubated simultaneously with an antibody-coated bead and the 125I-labeled antibody. Osteocalcin contained in the sample is immunologically bound by the immobilized antibody and the radiolabeled antibody to form a “sandwich” complex: bead/Anti-mouse osteocalcin, mouse osteocalcin, 125I-anti-mouse osteocalcin. At the end of the overnight incubation period, the bead was washed to remove any unbound labeled antibody and other components. The radioactivity bound to the bead was measured in a gamma counter. The radioactivity of the bound antibody complex is directly proportional to the amount of mouse osteocalcin in the sample. Because the amount of extracellular matrix proteins interferes with total cellular protein determination, the data were determined and expressed (as ng/mL) for each culture dish.
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Figure 1. Alizarin Red S stain of the nodules. Nodule cultures were stained with 2% Alizarin Red S calcium stain at the end of the experimental period (day 30). Treatments of control incubations are shown on the left column (A, D); glucose in the middle (B, E), and mannitol on the right (C, F). Macroscopic picture of a representative culture dish is shown on top row (A–C) and 25⫻ magnification in the bottom row (D–F). Glucose significantly increases the number and size of bone nodules.
Alkaline Phosphatase Alkaline phosphatase enzyme activity in the medium was determined by measuring the hydrolysis of p-nitrophenyl phosphate according to the manufacturer’s instructions (Sigma, St. Louis, MO) as previously described.1,2,36 –38 Medium was incubated with 30 mmol/L p-nitrophenyl phosphate in an alkaline buffer (glycine, pH 10.5) for 60 min in a 37°C water bath. The reaction was terminated by adding 2 mL of ice-cold 0.1 N NaOH. The amount of pnitrophenyl phosphate released was measured at 410 nm using the Spectramax 250 microplate reader (Molecular Dynamics, Sunnyvale, CA). Because the amount of extracellular matrix proteins interferes with total cellular protein determination, the data have been determined and expressed as alkaline phosphatase enzyme activity per culture dish (in pmol) of p-nitrophenol. Statistical Analysis Test for significance of difference, between-group means was performed using analysis of variance with a post-priori multiple comparison. All calculations were performed using the statistical analysis package SYSTAT (SYSTAT Evanston, IL). A p value ⬍ 0.05 was considered significant. Results Nodule Formation and Distribution At the end of the experimental period (day 30), culture dishes were stained with the Alizarin Red S calcium stain. Cells incu-
bated in control (Figure 1A, D) or mannitol (Figure 1C, F) -supplemented medium developed nodules with a regular pattern: larger calcifying foci surrounded by smaller foci in different stages of calcification. On the other hand, cells incubated in the glucose-supplemented medium developed much larger nodules that were irregularly scattered throughout the culture dish (Figure 1B, E). Total number of nodules per culture dish was quantified macroscopically. Compared with both the control and mannitolsupplemented medium, glucose supplementation significantly increased the number of nodules (Figure 2). Nodular structure could be better appreciated when examined under high magnification (25⫻; Figure 1D–F). Incubation with the glucose-supplemented medium (Figure 1E) resulted in nodules greater than four times the maximum size of nodules formed in either the control (Figure 1D) or mannitol (Figure 1F) -supplemented medium. In contrast to the rounded shape and uniform calcification pattern of the nodules formed in either the control (Figure 1D) or mannitol (Figure 1F) -supplemented medium, incubation with glucose resulted in irregular shape and pattern of calcification (Figure 1E). To account for the irregular nodular shape and calcification pattern observed in the glucose-treated cells, the total and calcified area of large nodules (area ⬎ 0.1 mm2) was determined. The total area, which reflects the entire area of the nodule, was plotted against the calcified area, defined as the dark brownblack stained area. Compared with both control and mannitoltreated cells, glucose significantly increased both the total and calcified nodular area (Figure 3). Total and calcified area of nodules formed in the presence of mannitol was not different
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Figure 2. Total number of nodules per culture dish. Total number of nodules per culture dish was determined macroscopically from the Alizarin Red S-stained culture dishes, day 30 (n⫽6 per treatment from two independent experiments). Glucose significantly increases the number of nodules compared with both control and mannitol. Data are expressed as mean ⫾ SEM. *Different than control and mannitol; p ⬍ 0.05.
from that of control. Furthermore, glucose also significantly increased the uncalcified nodular area compared with both control and mannitol treatment (data not shown). On the other hand, the percentage of calcified area relative to total nodular area was similar among all the treatment groups (data not shown). Large nodules develop from small calcifying foci; thus, the effect of glucose on the formation of the small calcifying areas devoid of discrete nodules were quantified at 50⫻ magnification. Compared with both control and mannitol treatment, glucose significantly increased the number of small nodules (Figure 4). In the presence of mannitol, the number of small nodules was similar to that of control. Calcium Uptake To monitor nodule development, calcium uptake by the cell cultures was determined every 48 –72 h. During the initial 2 weeks of incubation, there was minimal uptake of calcium by the cells (proliferation phase; Figure 5). At around day 16 –18, significant calcium uptake occurs (matrix maturation and calcification) and increases for the duration of the incubation period.
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Figure 4. Number of small nodules per visual field. Total number of small nodules per visual field was counted (n⫽20 random areas per treatment group from two independent experiments). Glucose significantly increases the number of small nodules. Data are expressed as mean ⫾ SEM. *Different than control and mannitol; p ⬍ 0.05.
However, in the presence of glucose, the daily calcium uptake is significantly inhibited compared with both control and mannitol treatment (Figure 5). The daily calcium uptake was similar in the control and mannitol groups. Cumulative calcium uptake was determined by summing the daily uptake values for each of the culture dishes. During the first 2 weeks, there was no significant calcium uptake into the cultures (proliferation and early matrix development phase). In both control and mannitol-treated cultures, there was a rapid phase of calcium incorporation that began between days 16 and 18 (late matrix maturation phase; Figure 6). There was similar cumulative calcium uptake into both the control and mannitol-treated cells. In contrast, cumulative calcium uptake in the glucosetreated cultures was significantly inhibited from day 23 (matrix mineralization) and proceeded at a steady rate, as opposed to the exponential increase observed in the control and mannitol-treated cells (Figure 6). To further confirm the validity of the calculated cumulative calcium uptake results and the effect of treatment on calcium incorporation into the nodules, the total calcium uptake into the cultures was determined by acid digestion. At the end of the experimental period (day 30), randomly selected cultures were subjected to digestion with 2N HCl and acid-soluble calcium content was determined by automated fluorometric titration analysis. Cell cultures treated with glucose had significantly less calcium incorporation than those treated with control and mannitol (Figure 7). Total calcium uptake in the mannitol-treated cultures was similar to that of control incubations. The total calcium content of the cultures obtained by acid digestion correlated well with and confirmed the cumulative calcium uptake calculated using the daily calcium values (Figures 6, 7). Proliferation of MC 3T3-E1 Osteoblast-like Cells
Figure 3. Total and calcified area of the large nodules. Total and calcified area of nodules larger than 0.1 mm2 was determined under 25⫻ magnification. The presence of glucose significantly increases both total and calcified area of the nodules. Data are expressed as mean ⫾ SEM; n⫽20 random nodules per treatment group from two independent experiments. *Different than control and mannitol; p ⬍ 0.05.
At the end of the experimental period, DNA synthesis was determined by [3H]thymidine incorporation assay. Compared with both control and mannitol-treated cells, the presence of glucose significantly increased [3H]thymidine incorporation into the cultures. Thymidine uptake of the mannitol-treated cultures was similar to control (Figure 8). Osteocalcin Production and Alkaline Phosphatase Activity To investigate osteoblast function, osteocalcin content, and alkaline phosphatase activity, markers of osteoblast function were
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Figure 5. Daily calcium uptake of the nodules. Daily calcium uptake was determined every 48 –72 h. Glucose significantly inhibits calcium uptake into nodules. Data are expressed as means ⫾ SEM, n⫽8 per treatment group. *Different than control and mannitol; p ⬍ 0.05.
determined from the culture supernatant at day 30. There was no difference in the osteocalcin concentration of culture supernatant between any of the treatment groups (Figure 9). Compared with both control and mannitol-treated cells, the presence of glucose significantly increased alkaline phosphatase activity of the culture supernatant. However, alkaline phosphatase activity of the mannitol-treated cultures was similar to control (Figure 10). Discussion Clinical studies have suggested an effect of glucose on bone metabolism. Hypercalciuria and bone loss has been observed in patients with poorly controlled diabetes.19,30,43 Bone mineral content has been reported to be decreased in patients with type 1 diabetes and seems to correlate with the duration of diabetes.13,17,18,20,52 Based upon clinical observations, it has been suggested that the osteopenia associated with diabetes is the result of a decrease in bone formation rather than an increase of bone resorption.17,52 In a histomorphometric study of eight patients with diabetes and low bone mineral density, the bone formation rate was 24% of normal.16 In another histological
Figure 6. Cumulative calcium uptake of the nodules. Cumulative calcium uptake was determined by summing the daily uptake values for each of the culture dishes. Glucose significantly inhibits cumulative calcium uptake. Data are expressed as means ⫾ SEM, n⫽8 per treatment group. *Different than control and mannitol; p ⬍ 0.05.
study, Kelin and Frost reported that in patients with diabetes, the rate of osteon formation was decreased by 36% compared with normal.14 Although diabetes appears to have a negative effect on the bone mineral, data are limited and very little is known about potential mechanism(s). During development, preosteoblasts undergo a series of differentiation stages, such as proliferation, matrix development, and matrix mineralization. The gradual acquisition of osteoblast phenotype is well characterized by the sequential up- and downregulation of osteoblast-specific genes.40 The proliferation stage is characterized by minimal expression of markers of the mature osteoblast phenotype. The transition of proliferation to matrix differentiation stage is indicated by the upregulation of genes associated with matrix development and maturation, collagen and alkaline phosphatase being the earliest. With the expression of osteoblast specific markers, proliferation slows down and matrix deposition and matrix maturation take place. Alkaline phosphatase values reach a maximum during the matrix maturation stage and are downregulated during late stages of matrix mineralization. High osteocalcin and osteopontin values reflect the mineralization period. Calcium accumulation of the cultures
Figure 7. Total calcium uptake of the nodules. Total calcium per dish was determined by acid digestion of randomly selected cultures at day 30. Cultures treated with glucose had significantly lower total calcium uptake compared with both control and mannitol. Data are expressed as means ⫾ SEM, n⫽3 per treatment group. *Different than control and mannitol; p ⬍ 0.05.
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Figure 8. Cellular proliferation of MC 3T3 cells. At the end of the experimental period (day 30), DNA synthesis was determined by [3H]thymidine pulse labeling of the cultures. Glucose significantly increased thymidine uptake into the cultures, compared with both control and mannitol. Data are expressed as means ⫾ SEM, n⫽5 per treatment group. *Different than both control and mannitol; p ⬍ 0.05.
Figure 10. Alkaline phosphatase enzyme activity. At the end of the experimental period (day 30), alkaline phosphatase enzyme activity was determined from conditioned media. Glucose significantly increased alkaline phosphatase enzyme activity, compared with both control and mannitol. Data are expressed as means ⫾ SEM, n⫽9 per treatment group. *Different than both control and mannitol; p ⬍ 0.05.
starts in the matrix development phase and is maximal during mineralization. There is a reciprocal correlation between osteoblast proliferation and differentiation. Tissue-specific transcriptional switches repress proliferation and permit differentiation.34,39 In preosteoblasts, differentiation switches are turned off while cells proliferate.34,39 In turn, expression of the mature osteoblast phenotype results in suppressing proliferation.34,39 Elements of the nuclear matrix, chromatin structure, and promoter regulation actively participate in the regulation of osteoblast proliferation and differentiation.39 Numerous in vitro model systems are utilized to study the sequential events associated with the development of osteoblasts, and the MC3T3-E1 cell line is an excellent and extensively used model. After repeated subcultivations, the MC3T3-E1 cells retain competency for differentiation, which is similar to primary cultures of calvarial osteoblasts.28 This is the first report investigating the direct effect of glucose on osteoblast differentiation in vitro. In the glucosetreated cultures, the area of the average nodule is significantly larger, and the distribution of calcium within the nodule is irregular compared with both the control and mannitol-treated cells (Figure 1). The irregular morphology of the nodules and the perturbation of calcium deposition imply that the presence of
high glucose concentration alters osteoblastic bone formation. To control for the osmotic effect of glucose, parallel cultures were incubated in medium supplemented with iso-osmolar concentration of mannitol. Nodules formed from the cells incubated with mannitol were indistinguishable from those formed in the control medium, thus supporting the hypothesis that glucose directly affects osteoblasts independent of an osmotic effect. The presence of glucose significantly increased the size and number of calcifying foci (both small and large nodules; Figure 1); however, the total amount of calcium deposited (determined by cumulative calcium uptake and acid digestion; Figures 6, 7) was significantly reduced. The fact that glucose-treated nodules are significantly larger (Figure 1) is seemingly inconsistent with the significant inhibition of calcium uptake (Figures 5–7). The calcium data are reliable, as two independent determinations of calcium uptake, cumulative calcium uptake (Figure 6) and total calcium uptake (Figure 7), give comparable results. To understand these findings, one needs to consider the dual nature of osteoblast development: differentiation downregulates proliferation and visa versa. These findings are in accordance with the above hypothesis, because in cultures of late mineralization stage (day 30), an increased proliferation rate of glucose-treated cultures was observed compared with both the control and mannitoltreated cells (Figure 8). The normally developing control and mannitol-treated cultures were, as expected, fairly quiescent at this stage of late mineralization. However, in the presence of high glucose concentration (15 mmol/L), osteoblastic proliferation was preserved (Figure 8). Taking into consideration that osteoblast proliferation inhibits differentiation, the presence of glucose results in larger nodules (increased proliferation) with less calcium deposition (inhibited differentiation). Moreover, both calcified and uncalcified nodular areas are significantly larger in the presence of high glucose content media compared with both normal and mannitol-treated cultures (Figure 3). The fact that the percentage of calcified area relative to total nodular area is similar among the groups further supports the hypothesis that in the presence of high glucose concentration osteoblasts undergo differentiation but with a significant delay compared with control or mannitol-treated cells. Alkaline phosphatase is expressed early in the developing osteoblasts during the phase of matrix development, and downregulated in calcifying osteoblasts. After 30 days in culture, in the MC3T3-E1 cells incubated in the presence of high glucose concentration, we observed a significantly higher level of alka-
Figure 9. Osteocalcin production by the nodules. At the end of the incubation period (day 30), osteocalcin release was determined from conditioned media. The total amount of released osteocalcin was similar in the treatment groups. Data are expressed as means ⫾ SEM, n⫽9 per treatment group.
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line phosphatase activity compared with both control and mannitol-treated cultures. The most likely explanation is that the alkaline phosphatase enzyme activity was downregulated in the fully developed mineralizing control and mannitol-treated cells. In contrast, the glucose-treated cells were still actively proliferating while maintaining a less mature phenotype with higher levels of alkaline phosphatase expression. Osteocalcin, a marker of late osteoblast differentiation, is expressed by highly differentiated osteoblasts during the phase of mineralization.40 Of note, although both osteocalcin and alkaline phosphatase are generally considered markers of mature osteoblast phenotype, during osteoblasts development, alkaline phosphatase expression reaches peak values earlier than osteocalcin. In the present study, no alteration was detected in the amount of osteocalcin released into the culture medium, using either control, glucose, or mannitol treatment. It is possible that the peak of osteocalcin expression was missed, and osteocalcin expression was already downregulated in the normally developing control and mannitol cultures, while cultures treated with high concentration of glucose maintained a very early phenotype characterized with low osteocalcin and high alkaline phosphatase expression. An alternative explanation of the similar osteocalcin values among the culture groups is that the osteocalcin concentration detected in the culture medium reflects the total osteocalcin released from the cultures. Considering that the thymidine incorporation assay detected an almost twofold increase in the proliferation rate in the presence of glucose compared with both control and mannitol treatments, it is likely that the absolute amount of osteocalcin produced by individual cells was inhibited by approximately 50% in the presence of glucose. This fact further suggests that cellular differentiation is inhibited in the presence of high glucose concentrations and supports the hypothesis that glucose has a negative regulatory effect on osteoblast differentiation. Another possible explanation of our findings is that a subset of the MC3T3-E1 cells was stimulated to proliferate. Although the MC3T3-E1 cells were initially isolated from a single clone, it has been recently shown that limiting dilutions and extensive subculturing lead to the development of MC3T3-E1 subclones with distinct mineralization potential.41,46 However, it might not be the case in the present study, because the MC3T3-E1 cells were utilized from frozen stocks of very low passage numbers (passage five to seven); thus, a subclonal selection of the cell line is unlikely. Examination of multiple time points during the course of osteoblast development and/or various glucose concentrations should provide further insight regarding the direct effect of glucose on osteoblast differentiation. Clearly, more work is necessary to determine the precise mechanism of action of glucose on osteoblasts. Glucose concentrations used in the present study correspond to healthy individuals (control, 5.5 mmol/L glucose) and a level frequently occurring in patients with poorly controlled diabetes associated with glycated hemoglobin (HbA1c) levels greater than 10% (15 mmol/L glucose). During cellular proliferation and early phases of matrix formation, no significant amount of calcium is deposited into the extracellular matrix. Consequently, the cumulative calcium uptake values of the three treatment groups are superimposable until day 21 (Figure 6). On the other hand, in the phase of intensive matrix calcification (starting at day 23), the presence of high glucose concentration leads to a differential effect of the different treatment groups. The cumulative calcium uptake into the glucose-treated nodules occurs at a slow steady rate, as opposed to the exponential accumulation of calcium observed in the control cultures (Figure 6). In the presence of elevated glucose concentration, it takes significantly longer to deposit the same amount of calcium as in the control or mannitol-treated incubation. This correlates well with the clinical estimation, that compared with healthy individuals, it takes 8 to 10
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times longer to form an average osteon in diabetes.14,17 At the end of the experimental period (day 30), the total amount of calcium uptake into the glucose-treated nodules was almost half of control. Interestingly, Rosato et al. estimated that patients with HbA1c levels greater than 10% have a bone formation rate less than half of that observed in healthy individuals.31 Considering that the glucose concentration used in the present study is comparable with poorly controlled diabetes and likely associated with HbA1c over 10%, our findings correlate well with this estimation. The results presented here are in agreement with the clinical findings that serum osteocalcin concentration is reduced in patients with diabetes, and the incidence of adynamic bone disease is increased in patients with diabetes and renal failure.24,26,31 According to several clinical studies, improved glycemic control increases serum osteocalcin concentration and bone turnover in patients with diabetes.24,31 The hypercalciuria associated with uncontrolled diabetes can be ameliorated with better diabetic control.24,30 However, the mechanism is unknown, because the lowering of urinary calcium concentrations is independent from serum parathyroid hormone levels.24 Considering the results of the present study, demonstrating that high glucose concentration inhibits osteoblast maturation and calcium deposition into forming bone nodules, it is possible that better glycemic control might improve osteoblastic function and thus calcium deposition into bone. The improved osteoblastic mineralization with increased calcium deposition into bone would lower serum calcium concentrations and thus ameliorate the hypercalciuria. In summary, the present study is the first to demonstrate that elevated glucose concentration inhibits osteoblastic calcium deposition. Utilizing the in vitro bone nodule formation assay, we demonstrated that a glucose concentration similar to those observed in patients with poorly controlled diabetes causes significant inhibition of calcium deposition. Because there is a reciprocal correlation between osteoblast proliferation and maturation, we propose that in the presence of elevated glucose concentration, osteoblast proliferation is enhanced while the in vitro bone formation is significantly inhibited. The metabolic alterations in diabetes include hyperglycemia, metabolic acidosis, and insulin insufficiency. It is likely that these factors co-contribute to the development of diabetic bone disease. The present study demonstrates that glucose exhibits a profound inhibitory effect on osteoblastic calcium deposition. The direct osteoblast inhibitory effect of hyperglycemia might be a contributor to other manifestations of secondary osteoporosis, as hyperglycemia is a common side effect to glucocorticoid and immunosuppressive therapy.2,8,50 Understanding the effect of glucose on osteoblast differentiation and calcium deposition might provide a better understanding to the development and prevention of osteopenia associated with diabetes.
Acknowledgments: This work was supported, in part, by a Research Award from the American Diabetes Association (S. M. Sprague) and by the Soros Foundation, Budapest, Hungary (P. Szabo, no. 222/3/3688). This work was presented in preliminary form at the annual meeting of the American Society for Bone and Mineral Research, St Louis, MO, September 30 to October 4, 1999.
References 1. Balint, E., Marshall, C. F., and Sprague, S. M. Role of interleukin-6 in 2-microglubulin-induced bone mineral dissolution. Kidney Int 57:1599 –1607; 2000 2. Balint, E., Marshall, C. F., and Sprague, S. M. Effect of the vitamin D analogues paricalcitol and calcitriol on bone mineral in vitro. Am J Kidney Dis. 789 –796; 2000. 3. Buoillon, R. Diabetic bone disease. Calcif Tiss Int 49:155–160; 1991.
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4. Bushinsky, D. A., Krieger, N. S., Geisser, D. I., Grossman, E. B., and Coe, F. L. Effects of pH on bone calcium and proton fluxes in vitro. Am J Physiol 245:F204 –209; 1983. 5. Chiu, M. Y., Sprague, S. M., Bruce, D. S., Woodle, E. S., Thistlethwaite, J. R., and Josephson, M. A. Analysis of fracture prevalence in kidney-pancreas allograft recipients. J Am Soc Nephrol 9:677– 683; 1998. 6. Dawson, A. B. A note on the staining of the skeleton of cleared specimens with Alizarin Red S. Stain Technol 1:123–124; 1926. 7. Dempster, D. W. Bone histomorphometry in glucocorticoid-induced osteoporosis. J Bone Miner Res 4:137–141; 1989. 8. Epstein, S. Post-transplant bone disease: The role of immunosuppressive agents and the skeleton. J Bone Miner Res 11:1–7; 1996. 9. Gallagher, J. C., Melton, L. J., and Riggs, B. L. Examination of prevalence rates of possible factors in a population with a fracture of the proximal femur. Clin Orthop 153:158 –165; 1980. 10. Grotz, W. H., Mundinger, F. A., Gugel, B., Exner, V., Kirste, G., and Schollmeyer, P. J. Bone fracture and osteodensitomerty in kidney transplant recipients. Transplantation 58:912–915; 1994. 11. Haffner, S. M. and Bauer, R. L. The association of obesity and glucose and insulin concentrations with bone density in premenopausal and postmenopausal women. Metabolism 42:735–738; 1993. 12. Heath, H., Melton, L. J., and Chu, C. P. Diabetes mellitus and risk of skeletal fracture. N Engl J Med 303:567–570; 1980. 13. Kayath, M. J., Tavares, E. F., Dib, S. A., and Vieira, J. G. H. Prospective bone mineral density evaluation in patients with insulin-dependent diabetes mellitus. J Diab Compl 12:133–139; 1998. 14. Kelin, M. and Frost, H. M. The numbers of bone resorption and formation foci in rib. Henry Ford Hosp Med Bull 12:527–536; 1964. 15. Kelsey, J. L., Browner, W. S., Seeley, D. G., Nevitt, M. C., and Cummings, S. R. Risk factors for fractures of the distal forearm and proximal humerus. Am J Epidemiol 135:477– 489; 1992. 16. Krakauer, J. C., McKenna, M. J., Buderer, N. F., Rao, D. S., Whitehouse, F. W., and Parfitt, A. M. Bone loss and bone turnover in diabetes. Diabetes 44:775–782; 1995. 17. Levin, M. E., Boisseau, V. C., and Avioli, L. V. Effects of diabetes mellitus on bone mass in juvenile and adult-onset diabetes. N Engl J Med 294:241–244; 1976. 18. Mathiassen, B., Nielsen, S., Ditzel, J., and Rodbro, P. Long term bone loss in insulin-dependent diabetes mellitus. J Int Med 227:325–327; 1990. 19. McNair, P., Madsbad, S., Christensen, M. S., Christiansen, C., Faber, O. K., Binder, C., and Transbol, I. Bone mineral loss in insulin-treated diabetes mellitus: studies on pathogenesis. Acta Endocrinol (Copenh) 90:463– 472; 1979. 20. McNair, P. Bone mineral metabolism in human type I (insulin dependent) diabetes mellitus. Dan Med Bull 35:109 –121; 1988. 21. Meyer, H. E., Tverdal, A., and Falch, J. A. Risk factors for hip fracture in middle-aged Norwegian women and men. Am J Epidemiol 137:1203–1211; 1993. 22. Morohoshi, M., Fujisawa, K., Uchimura, I., and Numano, F. Glucose dependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro. Diabetes 45:954 –959; 1996. 23. Nisbeth, U., Lindh, E., Ljunghall, S., Backman, U., and Fellstrom, B. Fracture frequency after kidney transplantation. Transpl Proc 26:1764; 1994. 24. Okazaki, R., Totsuka, Y., Hamano, K., Ajima, M., Miura, M., Hirota, Y., Hata, K., Fukumoto, S., and Matsumoto, T. Metabolic improvement of poorly controlled noninsulin-dependent diabetes mellitus decreases bone turnover. J Clin Endocrinol Metab 82:2915–2920; 1997. 25. Osther, P. J., Bollerslev, J., Hansen, A. B., Engel, K., and Kildeberg, P. Pathophysiology of incomplete renal tubular acidosis in recurrent stone formers: evidence of disturbed calcium, bone and citrate metabolism. Urol Res 21:169 –173; 1993. 26. Pei, Y., Hercz, G., Greenwood, C., Segre, G., Manuel, A., Saiphoo, C., Fenton, S., and Sherrard, D. Renal osteodystrophy in diabetic patients. Kidney Int 44:159 –164; 1993. 27. Pri-Chen, S., Pitaru, S., Lokiec, F., and Savion, N. Basic fibroblastic growth factor enhances the growth and expression of the osteogenic phenotpe of dexamethasone-treated human bone marrow-derived bone-like cells in culture. Bone 23:111–117; 1998. 28. Quarles, L. D., Yohai, D. A., Lever, L. W., Caton, R., and Wenstrup, R. J. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development. J Bone Miner Res 7:683– 692; 1992. 29. Ramsey-Goldman, R., Dunn, J. E., Dunlop, D. D., Stuart, F. P., Abecassis, M. M., Kaufman, D. B., Longman, C. B., Salinger, M. H., and Sprague, S. M. Increased risk of fracture in patients receiving solid organ transplants. J Bone Miner Res 14:456 – 463; 1999.
Bone Vol. 28, No. 1 January 2001:21–28 30. Raskin, P., Stevenson, M. R. M., Barilla, D. E., and Pak, C. C. The hypercalciuria of diabetes mellitus: its amelioration with insulin. Clin Endocrinol 9:329 –335; 1978. 31. Rosato, M. T., Schneider, S. H., and Shapses, S. A. Bone turnover and insulin-like growth factor I levels increase after improved glycemic control in noninsulin-dependent diabetes mellitus. Calcif Tiss Int 63:107–111; 1998. 32. Sambrook, P. N., Eisman, J. A., Pocock, N. A., and Jenkins, A. B. Serum insulin and bone density in normal subjects. J Rheumatol 15:1415–1417; 1988. 33. Sebastian, A., Harris, S. T., Ottaway, J. H., Todd, K. M., and Morris, R. C. Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate. N Engl J Med 330:1776 –1781; 1994. 34. Siddhanti, S. R., and Quarles, L. D. Molecular to pharmacologic control of osteoblast proliferation and differentiation. J Cell Biochem 55:310 –320; 1994. 35. Smythe, H. A. Osteoarthritis, insulin and bone density. J Rheumatol 14:91–93; 1987. 36. Sprague, S. M., and Bushinsky, D. A. Mechanism of aluminum-induced calcium efflux from cultured mouse calvariae. Am J Physiol 258:F583–F588; 1990. 37. Sprague, S. M., Krieger, N. S., and Bushinsky, D. A. Aluminium inhibits bone nodule formation and calcification in vitro. Am J Physiol 264:F882–F890; 1993. 38. Sprague, S. M., Krieger, N. S., and Bushinsky, D. A. Greater inhibition of in vitro bone mineralization with metabolic than respiratory acidosis. Kidney Int 46:1199 –1206; 1994. 39. Stein, G. S., and Lian, J. B. Molecular mechanisms mediating proliferationdifferentiation interrelationships during progressive development of the osteoblast phenotype: Update 1995. Endocrine Rev 4:290 –297; 1995. 40. Stein, G. S., Lian, J. B., Stein, J. L., van Wijnen, A. J., Frenkel, B., and Montecino, M. Mechanisms regulating osteoblast proliferation and differentiation. In: Bilezikien, J. P., Raisz, L. G., and Rodan, G. A., Eds. Principles of Bone Biology. London: Academic, 1996; 69 – 86. 41. Sudo, H., Kodama, H.-A., Amagai, Y., Yamamoto, S., and Kasai, S. In vitro differentiation and calcification of a new clonal osteogenc cell line from newborn mouse calvaria. J Cell Biol 96:191–198; 1983. 42. Tamayo, R., Goldman, J., Villanueva, A., Walczak, N., Whitehouse, F., and Parfitt, M. Bone mass and bone cell function in diabetes [Abstract]. Diabetes 30(Suppl. 1):31A; 1981. 43. Tenbaum, E. Ueber Kalkausscheidung durch den Harn bei Diabetes. Z Biol 33:379 – 403; 1896. 44. van Daele, P. L., Stolk, R. P., Burger, H., Algra, D., Grobbee, D. E., Hofman, A., Birkenhager, J. C., and Pols, H. A. Bone density in non-insulin-dependent diabetes mellitus: The Rotterdam Study. Ann Intern Med 122:409 – 414; 1995. 45. Verhaeghe, J., Van Herck, E., Van Bree, R., Van Assche, F. A., and Bouillon, R. Osteocalcin during the reproductive cycle in normal and diabetic rats. J. Endocr 120:143–151; 1989. 46. Wang, D., Christensen, K., Chawla, K., Xiao, G., Krebsbach, P. H., and Franceschi, R. T. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation and mineralization potential. J Bone Miner Res 14:893–903; 1999. 47. Weinstock, R. S., Goland, R. S., Shane, E., Clemens, T. L., Lindsay, R., and Bilezikian, J. P. Bone mineral density in women with type II diabetes mellitus. J Bone Miner Res 4:97–101; 1989. 48. Whang, K., Tsai, D. C., Nam, E. K., Aitken, M., Sprague, S. M., Patel, P. K., and Healy, K. E. Ectopic bone formation via rhBMP-2 delivery from porous bioabsorbable polymer scaffolds. J Biomed Mater Res 42:491– 499; 1998. 49. Whang, K., Healy, K. E., Elenz, D. R., Nam, E. K., Tsai, D. C., Thomas, C. H, Nuber, G. W., Glorieux, F. H., Travers, R., and Sprague, S. M. Engineering bone regeneration with bioabsorbable scaffolds with novel microarchitecture. Tissue Eng 5:35–51; 1999. 50. Williams, J. P., Blair, H. C., McDonald, J. M., McKenna, M. A., Jordan, S. E., Williford, J., and Hardy, R. W. Regulation of osteoclastic bone resorption by glucose. Biochem Biophys Res Commun 235:646 – 651; 1997. 51. Wise, J. E., Kolb, E. L., and Sauder, S. E. Effect of glycemic control on growth velocity in children with IDDM. Diabetes care 15:826 – 830; 1992. 52. Wiske, P. S., Wentworth, S. M., Norton, J. A., Epstein, S., and Johnson, C. C. Evaluation of bone mass and growth in young diabetics. Metabolism 31:848 – 854; 1982. 53. Yano, K., Wasnich, R. D., Vogel, V. M., and Heilbrun, L. K. Bone mineral measurements among middle-aged and elderly Japanese residents in Hawaii. Am J Epidemiol 119:751–764; 1984.
Date Received: March 21, 2000 Date Revised: August 31, 2000 Date Accepted: September 11, 2000
Glucose-induced Inhibition of In Vitro Bone Mineralization E. BALINT,1,2 P. SZABO,2 C. F. MARSHALL,2 and S. M. SPRAGUE1,2 1
Department of Medicine and the 2Research Institute, Evanston-Northwestern Healthcare, Northwestern University Medical School, Evanston, IL, USA
Introduction Patients with diabetes tend to have an increased incidence of osteopenia that may be related to hyperglycemia. However, little is known about how glucose may alter bone formation and osteoblast maturation. To determine whether glucose affects osteoblastic calcium deposition, MC3T3-E1 cells were incubated in media containing either a normal (5.5 mmol/L) or high glucose concentration (15 mmol/L) or mannitol (15 mmol/L), and bone nodule formation was examined. Net calcium flux was measured thrice weekly and cumulative calcium uptake was determined. Compared with control incubations, glucose significantly inhibited daily and cumulative calcium uptake into the nodules. At the time of matrix maturation, cultures undergo a rapid phase of increased calcium deposition; this was significantly inhibited by the presence of glucose. Total calcium uptake, determined by acid digestion, was also significantly inhibited by glucose. Area and number of nodules were quantitated at the end of the incubation period (day 30) by staining with Alizarin Red S calcium stain. Compared with both control and mannitoltreated cultures, the number of nodules was increased by incubation with glucose. Furthermore, both the average total nodular area and calcified nodular area of large nodules were increased by glucose. Cellular proliferation as well as the release of markers of osteoblast activity (osteocalcin and alkaline phosphatase) were determined at the end of the experimental period (day 30). Cellular proliferation and alkaline phosphatase activity was significantly increased in the presence of glucose, however, the release of osteocalcin into culture media was similar in all three groups. In conclusion, the present study shows that elevated glucose concentration present throughout the development of murine osteoblasts stimulates cellular proliferation while inhibiting calcium uptake. The result of glucose inhibition of calcium uptake suggests that bone could be structurally altered in diabetes. (Bone 28:21–28; 2001) © 2001 by Elsevier Science Inc. All rights reserved.
Osteopenia has been associated with diabetes, however, data demonstrating a causal relationship are limited and controversial. Bone mineral density in patients with both type I and type II diabetes has been reported to be decreased by greater than 10% compared with gender- and age-matched healthy persons.17,18,20 Although most investigators agree that osteopenia occurs in patients with type I diabetes, those with type II diabetes may have normal or increased bone density.11,16,18,44,47 In those with type I diabetes, the degree of osteopenia correlates with duration of diabetes.13,52 Long-term bone loss has been reported to be more severe among patients with type I diabetes compared with those with type II.16,18 This discrepancy in bone mass between those with type I and type II diabetes may be due, in part, to the obesity frequently associated with type II diabetes, as obesity is associated with increased bone density,11,53 thus masking the osteopenic affect of diabetes. Epidemiological studies evaluating the relationship between diabetes and fractures are also conflicting. An increased prevalence of diabetes has been described in patients with osteoporotic fractures,3,15,21 however, diabetes does not appear to be an independent risk for osteoporotic fracture.9,12 The tendency that patients with diabetes may have increased fracture rates is demonstrated among patients undergoing solid organ transplantation. Fractures occur in 10 –30% of solid organ transplant recipients.10,29 However, in those with preexisting diabetes, the incidence of having at least one fracture is greater than 45% after renal transplantation.23,29 In patients undergoing kidney pancreas transplantation, up to 50% were reported to sustain at least one fracture within 5 years of transplantation.5,29 Although clinical data concerning the role of diabetes in causing bone disease are conflicting, it is likely that diabetes alters bone metabolism. Tamayo and colleagues found that osteoblastic collagen synthesis was disturbed in diabetes.42 Serum osteocalcin concentrations, a marker of osteoblastic activity, were significantly lower in spontaneously diabetic BB rats compared with nondiabetic rats.45 Smythe and colleagues reported a positive correlation between hyperinsulinemia and bone density; however, others were unable to confirm such a correlation.11,32,35 Hyperglycemia is also associated with hypercalciuria19,20,30 and impairment of vitamin D metabolism,20 factors associated with bone loss. Metabolic acidosis, which frequently accompanies hyperglycemia, is associated with bone loss in vivo25,33 and induces bone resorption while inhibiting bone formation in vitro.4,38 Very little is known about the effect of hyperglycemia on bone metabolism. Clinical studies concerning the correlation of bone mass and glycemic control are controversial. Wise et al. reported that linear growth velocity is dependent on metabolic
Key Words: Glucose; Osteoblast; Calcium; Bone nodule; Mineralization.
Address for correspondence and reprints: Dr. Stuart M. Sprague, Evanston Northwestern Healthcare, Division of Nephrology, 2650 Ridge Avenue, Evanston, IL 60201. E-mail: [email protected] *E. Balint’s current address: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA; P. Szabo’s current address: 2nd Department of Medicine, Semmelweis University Medical School, Budapest, Hungary. © 2001 by Elsevier Science Inc. All rights reserved.
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control in children with type I diabetes mellitus.51 Rosato and colleagues reported an increase of bone turnover after improving metabolic control of patients with type II diabetes.31 However, Okazaki et al. showed a decrease of bone turnover after metabolic improvement of poorly controlled type II diabetes mellitus.24 McNair et al. reported a correlation between loss of bone mass and fasting blood glucose concentrations.19 However, others were unable to demonstrate a correlation between glycosylated hemoglobin and change in bone mass.17,42 Morohoshi and colleagues demonstrated that high concentration of glucose induces the production of the potent bone resorbing cytokines, interleukin-6 (IL-6) and tumor necrosis factor-␣ (TNF-␣), from human monocytes.22 Glucose has been shown to have a direct activating effect on osteoclasts and seems to be the principal energy source for osteoclastic bone resorption.50 Even less information is available concerning the effect of glucose on bone formation. Thus, the purpose of the present study was to determine the effect of prolonged elevated glucose concentrations on osteoblastic bone formation. Using the in vitro bone nodule formation assay, these studies demonstrated that elevated glucose concentrations resulted in decreased calcium uptake with defective nodular mineralization. Materials and Methods Bone Nodule Formation Assay Bone nodule formation was induced as previously described.37,38 Briefly, MC3T3-E1 mouse osteoblast-like cells were plated at a density of 4.5 ⫻ 104 cells/35-mm Petri dish and incubated in ␣-MEM (containing 5.5 mmol/L glucose) supplemented with 10% fetal bovine serum, 50 IU/mL potassium penicillin, and 50 g/mL streptomycin (Life Technologies, Gaithersberg, MD). Cells were grown until confluent, designated as day 0, and nodule formation was induced by the addition of 50 g/mL ascorbic acid and 10 mmol/L -glycerophosphate to the medium. The dishes were randomly divided into three treatment groups: vehicle (control), 15 mmol/L D-glucose (glucose), or 15 mol/L D-mannitol (osmotic control). The progress of osteoblast development was monitored by measuring calcium deposition into the cultures (daily calcium uptake). Media was changed every 48 –72 h and the calcium content of the conditioned media determined. A culture dish incubated without cells and subjected to similar media changes was used for baseline calcium determinations (medium blank). The experiment was carried out until the stage of maximal calcium influx (day 30), and randomly selected culture dishes were stained using Alizarin Red S calcium stain. At the same time (day 30), another set of randomly selected culture dishes were subjected to acid digestion in order to assess the amount of total calcium content per culture dish. For osteocalcin and alkaline phosphatase determinations, conditioned media were collected and stored at ⫺70°C until analysis. Nodule Quantification At the end of the experimental period (day 30), randomly selected nodule culture dishes were stained for calcium with Alizarin Red S (Aldrich Chemical Company Inc., Milwaukee, WI) as previously described.6 Briefly, nodules were fixed in 10% buffered formalin, washed with distilled water, and stained with 2% Alizarin Red S for 2 min. Staining with Alizarin Red S is a standard method for visualization of nodular pattern and calcium deposition of osteoblast cultures in vitro.27 Alizarin Red S stains calcium dark red and the tissue yellow. The culture dish incubated without cells and subjected to similar media changes did not stain positive for calcium with Alizarin Red S (medium blank
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as negative control; data not shown). The number of calcifying foci per culture dish was counted macroscopically on a 5-mm grid. Culture dishes were also examined under 25⫻ and 50⫻ magnification to microscopically assess nodule number, calcified, and uncalcified area. The number and area of calcified foci was determined by video analysis using the Osteomeasure histomorphometric software (OsteoMetrics Inc., Decatur, GA) as previously described.48,49 Large nodules include calcifying foci larger than 0.1 mm2, and small nodules are calcifying areas smaller than 0.1 mm2. Total and calcified area of large nodules was determined from randomly selected culture dishes from two independent experiments (n⫽20). Number of small nodules was determined at 50⫻ magnification from 20 randomly selected visual fields from two independent experiments (n⫽20). Calcium Calcium concentrations were determined from conditioned media immediately after collection using automated fluorometric titration analysis (Calcette; Precision Systems, Sudbury, MA) as previously described.1,37,38 Daily calcium uptake of the cultures was calculated by subtracting the value of conditioned media for each culture dish from the value of medium blank. A positive flux (influx) indicates calcium uptake from the medium into the cultures. Cumulative calcium uptake values were calculated by summing the daily uptake values for each culture dish. Total calcium uptake was determined from randomly selected culture dishes at day 30 using automated fluorometric titration analysis after an overnight incubation with 2 mL of 2 N HCl.1,37,38 Proliferation Assay On day 30, DNA synthesis was measured by pulse labeling the cultures with 10 Ci of 3H-labeled thymidine (DuPont NEN, Boston, MA) for 2 h as previously described.37 At the end of the incubation period, medium was aspirated and cells washed in PBS, then incubated in 5% trichloroacetic acid (TCA) for 20 min. The cells were then passed through glass-fiber filters that had been presoaked with 5% TCA. Filters were then washed with 95% ethanol, dried overnight, and placed into 10 mL of liquid scintillation cocktail (EcoLume; ICN, Costa Mesa, CA). Radioactivity of the filters was determined by liquid scintillation spectrometry. Because the amount of extracellular matrix proteins interferes with total cellular protein determination, the data were determined as total 3H-labeled thymidine incorporation per culture dish and expressed as percentage of control. Osteocalcin Osteocalcin content of the medium was measured using a mousespecific IRMA (Immutopics, Inc., San Clemente, CA). Briefly, sample containing mouse osteocalcin was incubated simultaneously with an antibody-coated bead and the 125I-labeled antibody. Osteocalcin contained in the sample is immunologically bound by the immobilized antibody and the radiolabeled antibody to form a “sandwich” complex: bead/Anti-mouse osteocalcin, mouse osteocalcin, 125I-anti-mouse osteocalcin. At the end of the overnight incubation period, the bead was washed to remove any unbound labeled antibody and other components. The radioactivity bound to the bead was measured in a gamma counter. The radioactivity of the bound antibody complex is directly proportional to the amount of mouse osteocalcin in the sample. Because the amount of extracellular matrix proteins interferes with total cellular protein determination, the data were determined and expressed (as ng/mL) for each culture dish.
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Figure 1. Alizarin Red S stain of the nodules. Nodule cultures were stained with 2% Alizarin Red S calcium stain at the end of the experimental period (day 30). Treatments of control incubations are shown on the left column (A, D); glucose in the middle (B, E), and mannitol on the right (C, F). Macroscopic picture of a representative culture dish is shown on top row (A–C) and 25⫻ magnification in the bottom row (D–F). Glucose significantly increases the number and size of bone nodules.
Alkaline Phosphatase Alkaline phosphatase enzyme activity in the medium was determined by measuring the hydrolysis of p-nitrophenyl phosphate according to the manufacturer’s instructions (Sigma, St. Louis, MO) as previously described.1,2,36 –38 Medium was incubated with 30 mmol/L p-nitrophenyl phosphate in an alkaline buffer (glycine, pH 10.5) for 60 min in a 37°C water bath. The reaction was terminated by adding 2 mL of ice-cold 0.1 N NaOH. The amount of pnitrophenyl phosphate released was measured at 410 nm using the Spectramax 250 microplate reader (Molecular Dynamics, Sunnyvale, CA). Because the amount of extracellular matrix proteins interferes with total cellular protein determination, the data have been determined and expressed as alkaline phosphatase enzyme activity per culture dish (in pmol) of p-nitrophenol. Statistical Analysis Test for significance of difference, between-group means was performed using analysis of variance with a post-priori multiple comparison. All calculations were performed using the statistical analysis package SYSTAT (SYSTAT Evanston, IL). A p value ⬍ 0.05 was considered significant. Results Nodule Formation and Distribution At the end of the experimental period (day 30), culture dishes were stained with the Alizarin Red S calcium stain. Cells incu-
bated in control (Figure 1A, D) or mannitol (Figure 1C, F) -supplemented medium developed nodules with a regular pattern: larger calcifying foci surrounded by smaller foci in different stages of calcification. On the other hand, cells incubated in the glucose-supplemented medium developed much larger nodules that were irregularly scattered throughout the culture dish (Figure 1B, E). Total number of nodules per culture dish was quantified macroscopically. Compared with both the control and mannitolsupplemented medium, glucose supplementation significantly increased the number of nodules (Figure 2). Nodular structure could be better appreciated when examined under high magnification (25⫻; Figure 1D–F). Incubation with the glucose-supplemented medium (Figure 1E) resulted in nodules greater than four times the maximum size of nodules formed in either the control (Figure 1D) or mannitol (Figure 1F) -supplemented medium. In contrast to the rounded shape and uniform calcification pattern of the nodules formed in either the control (Figure 1D) or mannitol (Figure 1F) -supplemented medium, incubation with glucose resulted in irregular shape and pattern of calcification (Figure 1E). To account for the irregular nodular shape and calcification pattern observed in the glucose-treated cells, the total and calcified area of large nodules (area ⬎ 0.1 mm2) was determined. The total area, which reflects the entire area of the nodule, was plotted against the calcified area, defined as the dark brownblack stained area. Compared with both control and mannitoltreated cells, glucose significantly increased both the total and calcified nodular area (Figure 3). Total and calcified area of nodules formed in the presence of mannitol was not different
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Figure 2. Total number of nodules per culture dish. Total number of nodules per culture dish was determined macroscopically from the Alizarin Red S-stained culture dishes, day 30 (n⫽6 per treatment from two independent experiments). Glucose significantly increases the number of nodules compared with both control and mannitol. Data are expressed as mean ⫾ SEM. *Different than control and mannitol; p ⬍ 0.05.
from that of control. Furthermore, glucose also significantly increased the uncalcified nodular area compared with both control and mannitol treatment (data not shown). On the other hand, the percentage of calcified area relative to total nodular area was similar among all the treatment groups (data not shown). Large nodules develop from small calcifying foci; thus, the effect of glucose on the formation of the small calcifying areas devoid of discrete nodules were quantified at 50⫻ magnification. Compared with both control and mannitol treatment, glucose significantly increased the number of small nodules (Figure 4). In the presence of mannitol, the number of small nodules was similar to that of control. Calcium Uptake To monitor nodule development, calcium uptake by the cell cultures was determined every 48 –72 h. During the initial 2 weeks of incubation, there was minimal uptake of calcium by the cells (proliferation phase; Figure 5). At around day 16 –18, significant calcium uptake occurs (matrix maturation and calcification) and increases for the duration of the incubation period.
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Figure 4. Number of small nodules per visual field. Total number of small nodules per visual field was counted (n⫽20 random areas per treatment group from two independent experiments). Glucose significantly increases the number of small nodules. Data are expressed as mean ⫾ SEM. *Different than control and mannitol; p ⬍ 0.05.
However, in the presence of glucose, the daily calcium uptake is significantly inhibited compared with both control and mannitol treatment (Figure 5). The daily calcium uptake was similar in the control and mannitol groups. Cumulative calcium uptake was determined by summing the daily uptake values for each of the culture dishes. During the first 2 weeks, there was no significant calcium uptake into the cultures (proliferation and early matrix development phase). In both control and mannitol-treated cultures, there was a rapid phase of calcium incorporation that began between days 16 and 18 (late matrix maturation phase; Figure 6). There was similar cumulative calcium uptake into both the control and mannitol-treated cells. In contrast, cumulative calcium uptake in the glucosetreated cultures was significantly inhibited from day 23 (matrix mineralization) and proceeded at a steady rate, as opposed to the exponential increase observed in the control and mannitol-treated cells (Figure 6). To further confirm the validity of the calculated cumulative calcium uptake results and the effect of treatment on calcium incorporation into the nodules, the total calcium uptake into the cultures was determined by acid digestion. At the end of the experimental period (day 30), randomly selected cultures were subjected to digestion with 2N HCl and acid-soluble calcium content was determined by automated fluorometric titration analysis. Cell cultures treated with glucose had significantly less calcium incorporation than those treated with control and mannitol (Figure 7). Total calcium uptake in the mannitol-treated cultures was similar to that of control incubations. The total calcium content of the cultures obtained by acid digestion correlated well with and confirmed the cumulative calcium uptake calculated using the daily calcium values (Figures 6, 7). Proliferation of MC 3T3-E1 Osteoblast-like Cells
Figure 3. Total and calcified area of the large nodules. Total and calcified area of nodules larger than 0.1 mm2 was determined under 25⫻ magnification. The presence of glucose significantly increases both total and calcified area of the nodules. Data are expressed as mean ⫾ SEM; n⫽20 random nodules per treatment group from two independent experiments. *Different than control and mannitol; p ⬍ 0.05.
At the end of the experimental period, DNA synthesis was determined by [3H]thymidine incorporation assay. Compared with both control and mannitol-treated cells, the presence of glucose significantly increased [3H]thymidine incorporation into the cultures. Thymidine uptake of the mannitol-treated cultures was similar to control (Figure 8). Osteocalcin Production and Alkaline Phosphatase Activity To investigate osteoblast function, osteocalcin content, and alkaline phosphatase activity, markers of osteoblast function were
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25
Figure 5. Daily calcium uptake of the nodules. Daily calcium uptake was determined every 48 –72 h. Glucose significantly inhibits calcium uptake into nodules. Data are expressed as means ⫾ SEM, n⫽8 per treatment group. *Different than control and mannitol; p ⬍ 0.05.
determined from the culture supernatant at day 30. There was no difference in the osteocalcin concentration of culture supernatant between any of the treatment groups (Figure 9). Compared with both control and mannitol-treated cells, the presence of glucose significantly increased alkaline phosphatase activity of the culture supernatant. However, alkaline phosphatase activity of the mannitol-treated cultures was similar to control (Figure 10). Discussion Clinical studies have suggested an effect of glucose on bone metabolism. Hypercalciuria and bone loss has been observed in patients with poorly controlled diabetes.19,30,43 Bone mineral content has been reported to be decreased in patients with type 1 diabetes and seems to correlate with the duration of diabetes.13,17,18,20,52 Based upon clinical observations, it has been suggested that the osteopenia associated with diabetes is the result of a decrease in bone formation rather than an increase of bone resorption.17,52 In a histomorphometric study of eight patients with diabetes and low bone mineral density, the bone formation rate was 24% of normal.16 In another histological
Figure 6. Cumulative calcium uptake of the nodules. Cumulative calcium uptake was determined by summing the daily uptake values for each of the culture dishes. Glucose significantly inhibits cumulative calcium uptake. Data are expressed as means ⫾ SEM, n⫽8 per treatment group. *Different than control and mannitol; p ⬍ 0.05.
study, Kelin and Frost reported that in patients with diabetes, the rate of osteon formation was decreased by 36% compared with normal.14 Although diabetes appears to have a negative effect on the bone mineral, data are limited and very little is known about potential mechanism(s). During development, preosteoblasts undergo a series of differentiation stages, such as proliferation, matrix development, and matrix mineralization. The gradual acquisition of osteoblast phenotype is well characterized by the sequential up- and downregulation of osteoblast-specific genes.40 The proliferation stage is characterized by minimal expression of markers of the mature osteoblast phenotype. The transition of proliferation to matrix differentiation stage is indicated by the upregulation of genes associated with matrix development and maturation, collagen and alkaline phosphatase being the earliest. With the expression of osteoblast specific markers, proliferation slows down and matrix deposition and matrix maturation take place. Alkaline phosphatase values reach a maximum during the matrix maturation stage and are downregulated during late stages of matrix mineralization. High osteocalcin and osteopontin values reflect the mineralization period. Calcium accumulation of the cultures
Figure 7. Total calcium uptake of the nodules. Total calcium per dish was determined by acid digestion of randomly selected cultures at day 30. Cultures treated with glucose had significantly lower total calcium uptake compared with both control and mannitol. Data are expressed as means ⫾ SEM, n⫽3 per treatment group. *Different than control and mannitol; p ⬍ 0.05.
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Figure 8. Cellular proliferation of MC 3T3 cells. At the end of the experimental period (day 30), DNA synthesis was determined by [3H]thymidine pulse labeling of the cultures. Glucose significantly increased thymidine uptake into the cultures, compared with both control and mannitol. Data are expressed as means ⫾ SEM, n⫽5 per treatment group. *Different than both control and mannitol; p ⬍ 0.05.
Figure 10. Alkaline phosphatase enzyme activity. At the end of the experimental period (day 30), alkaline phosphatase enzyme activity was determined from conditioned media. Glucose significantly increased alkaline phosphatase enzyme activity, compared with both control and mannitol. Data are expressed as means ⫾ SEM, n⫽9 per treatment group. *Different than both control and mannitol; p ⬍ 0.05.
starts in the matrix development phase and is maximal during mineralization. There is a reciprocal correlation between osteoblast proliferation and differentiation. Tissue-specific transcriptional switches repress proliferation and permit differentiation.34,39 In preosteoblasts, differentiation switches are turned off while cells proliferate.34,39 In turn, expression of the mature osteoblast phenotype results in suppressing proliferation.34,39 Elements of the nuclear matrix, chromatin structure, and promoter regulation actively participate in the regulation of osteoblast proliferation and differentiation.39 Numerous in vitro model systems are utilized to study the sequential events associated with the development of osteoblasts, and the MC3T3-E1 cell line is an excellent and extensively used model. After repeated subcultivations, the MC3T3-E1 cells retain competency for differentiation, which is similar to primary cultures of calvarial osteoblasts.28 This is the first report investigating the direct effect of glucose on osteoblast differentiation in vitro. In the glucosetreated cultures, the area of the average nodule is significantly larger, and the distribution of calcium within the nodule is irregular compared with both the control and mannitol-treated cells (Figure 1). The irregular morphology of the nodules and the perturbation of calcium deposition imply that the presence of
high glucose concentration alters osteoblastic bone formation. To control for the osmotic effect of glucose, parallel cultures were incubated in medium supplemented with iso-osmolar concentration of mannitol. Nodules formed from the cells incubated with mannitol were indistinguishable from those formed in the control medium, thus supporting the hypothesis that glucose directly affects osteoblasts independent of an osmotic effect. The presence of glucose significantly increased the size and number of calcifying foci (both small and large nodules; Figure 1); however, the total amount of calcium deposited (determined by cumulative calcium uptake and acid digestion; Figures 6, 7) was significantly reduced. The fact that glucose-treated nodules are significantly larger (Figure 1) is seemingly inconsistent with the significant inhibition of calcium uptake (Figures 5–7). The calcium data are reliable, as two independent determinations of calcium uptake, cumulative calcium uptake (Figure 6) and total calcium uptake (Figure 7), give comparable results. To understand these findings, one needs to consider the dual nature of osteoblast development: differentiation downregulates proliferation and visa versa. These findings are in accordance with the above hypothesis, because in cultures of late mineralization stage (day 30), an increased proliferation rate of glucose-treated cultures was observed compared with both the control and mannitoltreated cells (Figure 8). The normally developing control and mannitol-treated cultures were, as expected, fairly quiescent at this stage of late mineralization. However, in the presence of high glucose concentration (15 mmol/L), osteoblastic proliferation was preserved (Figure 8). Taking into consideration that osteoblast proliferation inhibits differentiation, the presence of glucose results in larger nodules (increased proliferation) with less calcium deposition (inhibited differentiation). Moreover, both calcified and uncalcified nodular areas are significantly larger in the presence of high glucose content media compared with both normal and mannitol-treated cultures (Figure 3). The fact that the percentage of calcified area relative to total nodular area is similar among the groups further supports the hypothesis that in the presence of high glucose concentration osteoblasts undergo differentiation but with a significant delay compared with control or mannitol-treated cells. Alkaline phosphatase is expressed early in the developing osteoblasts during the phase of matrix development, and downregulated in calcifying osteoblasts. After 30 days in culture, in the MC3T3-E1 cells incubated in the presence of high glucose concentration, we observed a significantly higher level of alka-
Figure 9. Osteocalcin production by the nodules. At the end of the incubation period (day 30), osteocalcin release was determined from conditioned media. The total amount of released osteocalcin was similar in the treatment groups. Data are expressed as means ⫾ SEM, n⫽9 per treatment group.
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line phosphatase activity compared with both control and mannitol-treated cultures. The most likely explanation is that the alkaline phosphatase enzyme activity was downregulated in the fully developed mineralizing control and mannitol-treated cells. In contrast, the glucose-treated cells were still actively proliferating while maintaining a less mature phenotype with higher levels of alkaline phosphatase expression. Osteocalcin, a marker of late osteoblast differentiation, is expressed by highly differentiated osteoblasts during the phase of mineralization.40 Of note, although both osteocalcin and alkaline phosphatase are generally considered markers of mature osteoblast phenotype, during osteoblasts development, alkaline phosphatase expression reaches peak values earlier than osteocalcin. In the present study, no alteration was detected in the amount of osteocalcin released into the culture medium, using either control, glucose, or mannitol treatment. It is possible that the peak of osteocalcin expression was missed, and osteocalcin expression was already downregulated in the normally developing control and mannitol cultures, while cultures treated with high concentration of glucose maintained a very early phenotype characterized with low osteocalcin and high alkaline phosphatase expression. An alternative explanation of the similar osteocalcin values among the culture groups is that the osteocalcin concentration detected in the culture medium reflects the total osteocalcin released from the cultures. Considering that the thymidine incorporation assay detected an almost twofold increase in the proliferation rate in the presence of glucose compared with both control and mannitol treatments, it is likely that the absolute amount of osteocalcin produced by individual cells was inhibited by approximately 50% in the presence of glucose. This fact further suggests that cellular differentiation is inhibited in the presence of high glucose concentrations and supports the hypothesis that glucose has a negative regulatory effect on osteoblast differentiation. Another possible explanation of our findings is that a subset of the MC3T3-E1 cells was stimulated to proliferate. Although the MC3T3-E1 cells were initially isolated from a single clone, it has been recently shown that limiting dilutions and extensive subculturing lead to the development of MC3T3-E1 subclones with distinct mineralization potential.41,46 However, it might not be the case in the present study, because the MC3T3-E1 cells were utilized from frozen stocks of very low passage numbers (passage five to seven); thus, a subclonal selection of the cell line is unlikely. Examination of multiple time points during the course of osteoblast development and/or various glucose concentrations should provide further insight regarding the direct effect of glucose on osteoblast differentiation. Clearly, more work is necessary to determine the precise mechanism of action of glucose on osteoblasts. Glucose concentrations used in the present study correspond to healthy individuals (control, 5.5 mmol/L glucose) and a level frequently occurring in patients with poorly controlled diabetes associated with glycated hemoglobin (HbA1c) levels greater than 10% (15 mmol/L glucose). During cellular proliferation and early phases of matrix formation, no significant amount of calcium is deposited into the extracellular matrix. Consequently, the cumulative calcium uptake values of the three treatment groups are superimposable until day 21 (Figure 6). On the other hand, in the phase of intensive matrix calcification (starting at day 23), the presence of high glucose concentration leads to a differential effect of the different treatment groups. The cumulative calcium uptake into the glucose-treated nodules occurs at a slow steady rate, as opposed to the exponential accumulation of calcium observed in the control cultures (Figure 6). In the presence of elevated glucose concentration, it takes significantly longer to deposit the same amount of calcium as in the control or mannitol-treated incubation. This correlates well with the clinical estimation, that compared with healthy individuals, it takes 8 to 10
E. Balint et al. Glucose and bone mineralization
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times longer to form an average osteon in diabetes.14,17 At the end of the experimental period (day 30), the total amount of calcium uptake into the glucose-treated nodules was almost half of control. Interestingly, Rosato et al. estimated that patients with HbA1c levels greater than 10% have a bone formation rate less than half of that observed in healthy individuals.31 Considering that the glucose concentration used in the present study is comparable with poorly controlled diabetes and likely associated with HbA1c over 10%, our findings correlate well with this estimation. The results presented here are in agreement with the clinical findings that serum osteocalcin concentration is reduced in patients with diabetes, and the incidence of adynamic bone disease is increased in patients with diabetes and renal failure.24,26,31 According to several clinical studies, improved glycemic control increases serum osteocalcin concentration and bone turnover in patients with diabetes.24,31 The hypercalciuria associated with uncontrolled diabetes can be ameliorated with better diabetic control.24,30 However, the mechanism is unknown, because the lowering of urinary calcium concentrations is independent from serum parathyroid hormone levels.24 Considering the results of the present study, demonstrating that high glucose concentration inhibits osteoblast maturation and calcium deposition into forming bone nodules, it is possible that better glycemic control might improve osteoblastic function and thus calcium deposition into bone. The improved osteoblastic mineralization with increased calcium deposition into bone would lower serum calcium concentrations and thus ameliorate the hypercalciuria. In summary, the present study is the first to demonstrate that elevated glucose concentration inhibits osteoblastic calcium deposition. Utilizing the in vitro bone nodule formation assay, we demonstrated that a glucose concentration similar to those observed in patients with poorly controlled diabetes causes significant inhibition of calcium deposition. Because there is a reciprocal correlation between osteoblast proliferation and maturation, we propose that in the presence of elevated glucose concentration, osteoblast proliferation is enhanced while the in vitro bone formation is significantly inhibited. The metabolic alterations in diabetes include hyperglycemia, metabolic acidosis, and insulin insufficiency. It is likely that these factors co-contribute to the development of diabetic bone disease. The present study demonstrates that glucose exhibits a profound inhibitory effect on osteoblastic calcium deposition. The direct osteoblast inhibitory effect of hyperglycemia might be a contributor to other manifestations of secondary osteoporosis, as hyperglycemia is a common side effect to glucocorticoid and immunosuppressive therapy.2,8,50 Understanding the effect of glucose on osteoblast differentiation and calcium deposition might provide a better understanding to the development and prevention of osteopenia associated with diabetes.
Acknowledgments: This work was supported, in part, by a Research Award from the American Diabetes Association (S. M. Sprague) and by the Soros Foundation, Budapest, Hungary (P. Szabo, no. 222/3/3688). This work was presented in preliminary form at the annual meeting of the American Society for Bone and Mineral Research, St Louis, MO, September 30 to October 4, 1999.
References 1. Balint, E., Marshall, C. F., and Sprague, S. M. Role of interleukin-6 in 2-microglubulin-induced bone mineral dissolution. Kidney Int 57:1599 –1607; 2000 2. Balint, E., Marshall, C. F., and Sprague, S. M. Effect of the vitamin D analogues paricalcitol and calcitriol on bone mineral in vitro. Am J Kidney Dis. 789 –796; 2000. 3. Buoillon, R. Diabetic bone disease. Calcif Tiss Int 49:155–160; 1991.
28
E. Balint et al. Glucose and bone mineralization
4. Bushinsky, D. A., Krieger, N. S., Geisser, D. I., Grossman, E. B., and Coe, F. L. Effects of pH on bone calcium and proton fluxes in vitro. Am J Physiol 245:F204 –209; 1983. 5. Chiu, M. Y., Sprague, S. M., Bruce, D. S., Woodle, E. S., Thistlethwaite, J. R., and Josephson, M. A. Analysis of fracture prevalence in kidney-pancreas allograft recipients. J Am Soc Nephrol 9:677– 683; 1998. 6. Dawson, A. B. A note on the staining of the skeleton of cleared specimens with Alizarin Red S. Stain Technol 1:123–124; 1926. 7. Dempster, D. W. Bone histomorphometry in glucocorticoid-induced osteoporosis. J Bone Miner Res 4:137–141; 1989. 8. Epstein, S. Post-transplant bone disease: The role of immunosuppressive agents and the skeleton. J Bone Miner Res 11:1–7; 1996. 9. Gallagher, J. C., Melton, L. J., and Riggs, B. L. Examination of prevalence rates of possible factors in a population with a fracture of the proximal femur. Clin Orthop 153:158 –165; 1980. 10. Grotz, W. H., Mundinger, F. A., Gugel, B., Exner, V., Kirste, G., and Schollmeyer, P. J. Bone fracture and osteodensitomerty in kidney transplant recipients. Transplantation 58:912–915; 1994. 11. Haffner, S. M. and Bauer, R. L. The association of obesity and glucose and insulin concentrations with bone density in premenopausal and postmenopausal women. Metabolism 42:735–738; 1993. 12. Heath, H., Melton, L. J., and Chu, C. P. Diabetes mellitus and risk of skeletal fracture. N Engl J Med 303:567–570; 1980. 13. Kayath, M. J., Tavares, E. F., Dib, S. A., and Vieira, J. G. H. Prospective bone mineral density evaluation in patients with insulin-dependent diabetes mellitus. J Diab Compl 12:133–139; 1998. 14. Kelin, M. and Frost, H. M. The numbers of bone resorption and formation foci in rib. Henry Ford Hosp Med Bull 12:527–536; 1964. 15. Kelsey, J. L., Browner, W. S., Seeley, D. G., Nevitt, M. C., and Cummings, S. R. Risk factors for fractures of the distal forearm and proximal humerus. Am J Epidemiol 135:477– 489; 1992. 16. Krakauer, J. C., McKenna, M. J., Buderer, N. F., Rao, D. S., Whitehouse, F. W., and Parfitt, A. M. Bone loss and bone turnover in diabetes. Diabetes 44:775–782; 1995. 17. Levin, M. E., Boisseau, V. C., and Avioli, L. V. Effects of diabetes mellitus on bone mass in juvenile and adult-onset diabetes. N Engl J Med 294:241–244; 1976. 18. Mathiassen, B., Nielsen, S., Ditzel, J., and Rodbro, P. Long term bone loss in insulin-dependent diabetes mellitus. J Int Med 227:325–327; 1990. 19. McNair, P., Madsbad, S., Christensen, M. S., Christiansen, C., Faber, O. K., Binder, C., and Transbol, I. Bone mineral loss in insulin-treated diabetes mellitus: studies on pathogenesis. Acta Endocrinol (Copenh) 90:463– 472; 1979. 20. McNair, P. Bone mineral metabolism in human type I (insulin dependent) diabetes mellitus. Dan Med Bull 35:109 –121; 1988. 21. Meyer, H. E., Tverdal, A., and Falch, J. A. Risk factors for hip fracture in middle-aged Norwegian women and men. Am J Epidemiol 137:1203–1211; 1993. 22. Morohoshi, M., Fujisawa, K., Uchimura, I., and Numano, F. Glucose dependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro. Diabetes 45:954 –959; 1996. 23. Nisbeth, U., Lindh, E., Ljunghall, S., Backman, U., and Fellstrom, B. Fracture frequency after kidney transplantation. Transpl Proc 26:1764; 1994. 24. Okazaki, R., Totsuka, Y., Hamano, K., Ajima, M., Miura, M., Hirota, Y., Hata, K., Fukumoto, S., and Matsumoto, T. Metabolic improvement of poorly controlled noninsulin-dependent diabetes mellitus decreases bone turnover. J Clin Endocrinol Metab 82:2915–2920; 1997. 25. Osther, P. J., Bollerslev, J., Hansen, A. B., Engel, K., and Kildeberg, P. Pathophysiology of incomplete renal tubular acidosis in recurrent stone formers: evidence of disturbed calcium, bone and citrate metabolism. Urol Res 21:169 –173; 1993. 26. Pei, Y., Hercz, G., Greenwood, C., Segre, G., Manuel, A., Saiphoo, C., Fenton, S., and Sherrard, D. Renal osteodystrophy in diabetic patients. Kidney Int 44:159 –164; 1993. 27. Pri-Chen, S., Pitaru, S., Lokiec, F., and Savion, N. Basic fibroblastic growth factor enhances the growth and expression of the osteogenic phenotpe of dexamethasone-treated human bone marrow-derived bone-like cells in culture. Bone 23:111–117; 1998. 28. Quarles, L. D., Yohai, D. A., Lever, L. W., Caton, R., and Wenstrup, R. J. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development. J Bone Miner Res 7:683– 692; 1992. 29. Ramsey-Goldman, R., Dunn, J. E., Dunlop, D. D., Stuart, F. P., Abecassis, M. M., Kaufman, D. B., Longman, C. B., Salinger, M. H., and Sprague, S. M. Increased risk of fracture in patients receiving solid organ transplants. J Bone Miner Res 14:456 – 463; 1999.
Bone Vol. 28, No. 1 January 2001:21–28 30. Raskin, P., Stevenson, M. R. M., Barilla, D. E., and Pak, C. C. The hypercalciuria of diabetes mellitus: its amelioration with insulin. Clin Endocrinol 9:329 –335; 1978. 31. Rosato, M. T., Schneider, S. H., and Shapses, S. A. Bone turnover and insulin-like growth factor I levels increase after improved glycemic control in noninsulin-dependent diabetes mellitus. Calcif Tiss Int 63:107–111; 1998. 32. Sambrook, P. N., Eisman, J. A., Pocock, N. A., and Jenkins, A. B. Serum insulin and bone density in normal subjects. J Rheumatol 15:1415–1417; 1988. 33. Sebastian, A., Harris, S. T., Ottaway, J. H., Todd, K. M., and Morris, R. C. Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate. N Engl J Med 330:1776 –1781; 1994. 34. Siddhanti, S. R., and Quarles, L. D. Molecular to pharmacologic control of osteoblast proliferation and differentiation. J Cell Biochem 55:310 –320; 1994. 35. Smythe, H. A. Osteoarthritis, insulin and bone density. J Rheumatol 14:91–93; 1987. 36. Sprague, S. M., and Bushinsky, D. A. Mechanism of aluminum-induced calcium efflux from cultured mouse calvariae. Am J Physiol 258:F583–F588; 1990. 37. Sprague, S. M., Krieger, N. S., and Bushinsky, D. A. Aluminium inhibits bone nodule formation and calcification in vitro. Am J Physiol 264:F882–F890; 1993. 38. Sprague, S. M., Krieger, N. S., and Bushinsky, D. A. Greater inhibition of in vitro bone mineralization with metabolic than respiratory acidosis. Kidney Int 46:1199 –1206; 1994. 39. Stein, G. S., and Lian, J. B. Molecular mechanisms mediating proliferationdifferentiation interrelationships during progressive development of the osteoblast phenotype: Update 1995. Endocrine Rev 4:290 –297; 1995. 40. Stein, G. S., Lian, J. B., Stein, J. L., van Wijnen, A. J., Frenkel, B., and Montecino, M. Mechanisms regulating osteoblast proliferation and differentiation. In: Bilezikien, J. P., Raisz, L. G., and Rodan, G. A., Eds. Principles of Bone Biology. London: Academic, 1996; 69 – 86. 41. Sudo, H., Kodama, H.-A., Amagai, Y., Yamamoto, S., and Kasai, S. In vitro differentiation and calcification of a new clonal osteogenc cell line from newborn mouse calvaria. J Cell Biol 96:191–198; 1983. 42. Tamayo, R., Goldman, J., Villanueva, A., Walczak, N., Whitehouse, F., and Parfitt, M. Bone mass and bone cell function in diabetes [Abstract]. Diabetes 30(Suppl. 1):31A; 1981. 43. Tenbaum, E. Ueber Kalkausscheidung durch den Harn bei Diabetes. Z Biol 33:379 – 403; 1896. 44. van Daele, P. L., Stolk, R. P., Burger, H., Algra, D., Grobbee, D. E., Hofman, A., Birkenhager, J. C., and Pols, H. A. Bone density in non-insulin-dependent diabetes mellitus: The Rotterdam Study. Ann Intern Med 122:409 – 414; 1995. 45. Verhaeghe, J., Van Herck, E., Van Bree, R., Van Assche, F. A., and Bouillon, R. Osteocalcin during the reproductive cycle in normal and diabetic rats. J. Endocr 120:143–151; 1989. 46. Wang, D., Christensen, K., Chawla, K., Xiao, G., Krebsbach, P. H., and Franceschi, R. T. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation and mineralization potential. J Bone Miner Res 14:893–903; 1999. 47. Weinstock, R. S., Goland, R. S., Shane, E., Clemens, T. L., Lindsay, R., and Bilezikian, J. P. Bone mineral density in women with type II diabetes mellitus. J Bone Miner Res 4:97–101; 1989. 48. Whang, K., Tsai, D. C., Nam, E. K., Aitken, M., Sprague, S. M., Patel, P. K., and Healy, K. E. Ectopic bone formation via rhBMP-2 delivery from porous bioabsorbable polymer scaffolds. J Biomed Mater Res 42:491– 499; 1998. 49. Whang, K., Healy, K. E., Elenz, D. R., Nam, E. K., Tsai, D. C., Thomas, C. H, Nuber, G. W., Glorieux, F. H., Travers, R., and Sprague, S. M. Engineering bone regeneration with bioabsorbable scaffolds with novel microarchitecture. Tissue Eng 5:35–51; 1999. 50. Williams, J. P., Blair, H. C., McDonald, J. M., McKenna, M. A., Jordan, S. E., Williford, J., and Hardy, R. W. Regulation of osteoclastic bone resorption by glucose. Biochem Biophys Res Commun 235:646 – 651; 1997. 51. Wise, J. E., Kolb, E. L., and Sauder, S. E. Effect of glycemic control on growth velocity in children with IDDM. Diabetes care 15:826 – 830; 1992. 52. Wiske, P. S., Wentworth, S. M., Norton, J. A., Epstein, S., and Johnson, C. C. Evaluation of bone mass and growth in young diabetics. Metabolism 31:848 – 854; 1982. 53. Yano, K., Wasnich, R. D., Vogel, V. M., and Heilbrun, L. K. Bone mineral measurements among middle-aged and elderly Japanese residents in Hawaii. Am J Epidemiol 119:751–764; 1984.
Date Received: March 21, 2000 Date Revised: August 31, 2000 Date Accepted: September 11, 2000