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Endocrinology Vol. 139, No. 1 51-56
Copyright © 1998 by The Endocrine Society

ARTICLES

Insulin Stimulates Vitamin C Recycling and Ascorbate Accumulation in Osteoblastic Cells1

Sami Qutob, S. Jeffrey Dixon and John X. Wilson

Department of Physiology and Division of Oral Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1

Address all correspondence and requests for reprints to: Dr. John X. Wilson, Department of Physiology, University of Western Ontario, London, Ontario, Canada N6A 5C1. E-mail: jwilson{at}physiology.uwo.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin modulates the differentiation and synthetic activity of osteoblasts, but its mechanisms of action are not fully understood. Because ascorbate also influences osteoblast differentiation and is a cofactor for collagen synthesis, we examined the effects of insulin on the transport and metabolism of vitamin C in osteoblastic cells. UMR-106 rat osteoblast-like cells accumulated ascorbate intracellularly when incubated with dehydroascorbic acid (DHAA; oxidized vitamin C). Insulin increased the intracellular concentration of ascorbate derived from DHAA and also increased the initial rates of uptake of DHAA and 2-deoxyglucose, but not that of ascorbate. A half-maximal effect on DHAA uptake was observed with approximately 100 pM insulin, whereas insulin-like growth factor I (IGF-I) was less potent. Preincubation with insulin for 6–12 h was required for stimulation, similar to the period needed for increased expression of facilitative hexose transporters (GLUT). DHAA uptake was inhibited by the GLUT antagonist cytochalasin B as well as by the GLUT substrates D-glucose and 2-deoxyglucose, whereas L-glucose and fructose had no effect. We conclude that insulin and IGF-I stimulate osteoblastic uptake of DHAA through facilitative hexose transporters. The relative potency of insulin in stimulating DHAA uptake is consistent with mediation by insulin receptors. DHAA is reduced to ascorbate within osteoblasts, maintaining a high intracellular concentration of ascorbate available for collagen synthesis. Impaired uptake of DHAA may contribute to the osteopenia associated with type I diabetes. In addition, cytotoxic levels of DHAA may accumulate in the extracellular fluid due to decreased transport activity and competitive inhibition by elevated concentrations of glucose.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN and insulin-like growth factor I (IGF-I) stimulate osteoblast differentiation, collagen synthesis, and bone formation (1, 2, 3, 4). For instance, local injection of insulin over the calvaria of adult mice directly stimulates bone formation (4). Normal collagen synthesis requires high concentrations of intracellular ascorbate (5).

Bone growth and remodeling are decreased in insulin-dependent (type I) diabetes mellitus, leading to osteopenia and osteoporosis (6, 7). On the other hand, patients with hyperinsulinemia, with or without hyperglycemia, have increased bone mineral density (8). There also is evidence of defective handling of vitamin C in diabetes (9, 10, 11, 12, 13). Localized scarcities of ascorbate might occur in insulin-sensitive tissues under diabetic conditions. For example, Chorvathova and Ginter (11) reported that vitamin C levels are subnormal in tissues of rats with insulin-dependent diabetes. The ascorbate content of mononuclear leukocytes is decreased in patients with insulin-dependent diabetes mellitus even when they consume normally adequate amounts of dietary vitamin C (13). Plasma concentrations of reduced vitamin C (ascorbate) are decreased and those of oxidized vitamin C (dehydroascorbic acid, DHAA) are elevated in some insulin-dependent diabetic patients (14, 15, 16). This may reflect oxidative stress, which contributes to the development of complications in diabetes (17). The redox state of vitamin C is important because at high concentrations, DHAA exerts direct cytotoxic (18, 19, 20) and lethal (21) effects.

Ascorbate influences the differentiation of preosteoblasts and is required for the synthesis of osteoid by mature osteoblasts (5, 22, 23). Interestingly, Hammarstrom (24) found that radioactivity from [14C]DHAA injected systemically into immature rats rapidly accumulated to persistently high levels in cartilage and bone at sites where mineralization was occurring. The purpose of the present study was to investigate the influence of insulin on the transport and metabolism of ascorbate and DHAA in osteoblastic cells. The UMR-106 cell line was chosen as an in vitro model of osteoblastic function. UMR-106 cells resemble other osteoblastic models in being incapable of de novo synthesis of ascorbate from glucose and in possessing a Na+-ascorbate cotransport system (25, 26). Transforming growth factor-ß increases the intracellular ascorbate concentration in UMR-106 cells by stimulating Na+-ascorbate cotransport (26). Additionally, these cells express high affinity insulin receptors (27, 28) that are clearly functional insofar as they have been shown to regulate both Na+-dependent phosphate transport (29) and glucose uptake mediated by facilitative hexose transporters (GLUT) (30, 31).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
{alpha}MEM and heat-inactivated FBS were obtained from Life Technologies (Burlington, Canada). L-[14C]Ascorbate (7 mCi/mmol) and 2-deoxy-D-[1,2-N-3H]glucose (26 Ci/mmol) were purchased from Dupont Canada (Lachine, Canada). Bovine albumin, crystallized, was obtained from ICN Biomedicals (Costa Mesa, CA). L-Ascorbic acid, ascorbic acid oxidase (EC 1.10.3.3), cycloheximide, 2-deoxyglucose, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), D,L-homocysteine, cytochalasin B, human recombinant IGF-I, and porcine insulin were obtained from Sigma Chemical Co. (St. Louis, MO). Insulin was dissolved in PBS containing 0.1 M NaOH and was prepared fresh daily. IGF-I was dissolved in PBS containing 10% acetic acid and was stored in aliquots at -80 C.

Cells and cell culture
The clonal osteosarcoma cell line UMR-106 (American Type Culture Collection, Rockville, MD) was subcultured twice weekly in {alpha}MEM supplemented with 10% FBS. The concentration of ascorbate in this medium was measured using HPLC with electrochemical detection (HPLC-ED) and was found to be less than 0.5 µM (i.e. ascorbate-free). Three days before uptake measurements, cells were seeded at a density of 104 cells/cm2 into 60-mm culture dishes. After 2 days, the medium was changed to serum-free {alpha}MEM with bovine albumin (1 mg/ml). Cultures were treated with insulin, IGF-I, or vehicle for the period indicated, before assay of transport activity or ascorbate concentration. In experiments that tested whether protein synthesis was required for stimulation of transport, cycloheximide (10 µM) was added at the same time as insulin. Cell morphology was examined by phase contrast microscopy.

Experimental procedures
Glucose-free transport medium consisted of 134 mM NaCl, 5.2 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 10 mM glucose, and 20 mM HEPES, adjusted to pH 7.3 with NaOH. The final Na+ concentration of this medium was 138 mM, and its osmolality was 300 mosmol/liter. pH was measured using a pH-sensitive electrode, osmolality was measured by freezing point depression, and the Na+ concentration was determined by flame photometry.

2-Deoxy-D-[3H]glucose transport studies were carried out in glucose-free transport medium for 1 min at 23 C (32). The concentration of radiolabeled 2-deoxyglucose was 60 µM (SA was adjusted with unlabeled 2-deoxyglucose to 3.3 mCi/mmol). Where indicated, cytochalasin B (10 µM) was added to inhibit facilitative hexose transporters.

Ascorbate was dissolved in ice-cold medium (vehicle) at the beginning of each experiment. To prevent nonenzymatic oxidation of [14C]ascorbate, stock solutions were dissolved in 3 mM aqueous homocysteine, fractionated into aliquots, and stored at -80 C. Before incubation with cells, the specific activity of [14C]ascorbate was adjusted by the addition of unlabeled ascorbate and was checked by HPLC-ED and scintillation counting seriatim (see below).

To measure the initial rate of ascorbate uptake, cells were washed and incubated with [14C]ascorbate (10 µM; 7 mCi/mmol) at 37 C. [14C]Ascorbate uptake by UMR-106 cells proceeded as a linear function of time for at least 1 min. Therefore, initial rates of uptake were determined using 1-min incubations with radiolabeled ascorbate.

Aliquots of incubation buffer were collected at the end of each uptake incubation. Incubations were terminated by washing cultures with ice-cold isoosmotic Tris-sucrose solution. The effectiveness of the washing procedure was confirmed by the observation that adding 100 µM unlabeled ascorbate to selected cultures and immediately removing it by washing at 37 C (nominally zero time exposure to the vitamin) did not alter the initial rate of [14C]ascorbate uptake during the subsequent transport assay.

Cells were harvested by osmotic lysis (1 ml water/dish) and mechanical scraping. An aliquot of the cell harvest was used for protein measurement (33), and the remainder was combined with scintillation cocktail. The radioactive contents of the buffer and cells were measured by liquid scintillation counting. Uptake rates were computed based on the specific activity of radiolabeled ascorbate in the medium and expressed as nanomoles of [14C]ascorbate per g cell protein/min.

[14C]DHAA was prepared by incubating [14C]ascorbate with ascorbic acid oxidase (1 U/ml) at 37 C for 1 min. Complete oxidation of ascorbate was verified using HPLC-ED. Specific activity was adjusted by adding unlabeled DHAA. Ascorbate concentrations in cells, medium, and incubation buffer were assayed by acidic extraction and HPLC-ED, according to a procedure described previously (26). Ascorbate was quantified with a Waters M460 amperometric detector (Waters Associates, Milford, MA). Representative chromatograms have been previously published (26). Identification of ascorbate was confirmed by its susceptibility to ascorbate oxidase. The ascorbate concentrations of experimental samples were determined by interpolation on an external standard curve.

Statistics
Results are presented as the mean ± SEM of n independent experiments, with either duplicate or triplicate replications in each experiment. In the figures, error bars were omitted when the SEM was less than the size of the symbol. Comparisons between mean values based on a single level of treatment were evaluated using paired t test. For simultaneous comparisons of two or more treatments, ANOVA and the Tukey-Kramer test evaluated differences between means. For all statistical tests, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have previously shown that UMR-106 rat osteoblastic cells accumulate intracellular ascorbate to a steady state concentration of 1 mM when incubated with ascorbate for 6 h (26). These cells achieved a comparable intracellular concentration of authentic ascorbate when incubated with 100 µM DHAA for only 10 min (Fig. 1Go). This means that the osteoblastic cells took up DHAA and reduced it to ascorbate, leading to the rapid accumulation of intracellular ascorbate even when extracellular ascorbate was absent. Pretreatment of cells with insulin (10 nM; 24 h) stimulated the uptake of DHAA and its reduction to ascorbate, indicated by a 45 ± 10% increase in intracellular accumulation of reduced vitamin C (Fig. 1Go). However, the percentage of intracellular vitamin C that was ascorbate remained unchanged: vehicle, 60 ± 10%; insulin, 56 ± 8% (n = 6).



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  		Figure 1. Insulin stimulates uptake and reduction of DHAA to ascorbate. UMR-106 cells were pretreated for 24 h with vehicle (control) or insulin (10 nM) in serum-free medium. They were then incubated with 100 µM [14C]DHAA for 10 min at 37 C. The intracellular concentration of total (reduced and oxidized) vitamin C was measured by scintillation counting, and ascorbate was measured by HPLC-ED. Plotted are the mean ± SEM from six independent experiments. *, P < 0.05 for effect of insulin compared with the relevant control value.

 
Transport activity was assessed by measuring the uptake of radiolabeled DHAA (10 µM; 23 C). Uptake was linear with time for at least 2 min (Fig. 2Go). Pretreatment of cells with insulin (10 nM; 24 h) increased the initial rate of DHAA uptake (Fig. 2Go). In a series of experiments, after 24-h treatment with vehicle or insulin (10 nM), the rates of DHAA uptake (10 µM; 23 C), expressed as nanomoles per g cell protein/min, were: vehicle, 66 ± 5; and insulin, 162 ± 16 (n = 25; P < 0.001). Thus, the mechanism by which insulin stimulates vitamin C recycling may include acceleration of DHAA transport activity.



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  		Figure 2. Insulin increases the initial rate of DHAA uptake. UMR-106 cells were pretreated for 24 h with vehicle (control) or insulin (10 nM) in serum-free medium. They were then incubated with 10 µM [14C]DHAA for the indicated periods at 23 C. Plotted are the mean ± SEM from three independent experiments.

 
Subsequent experiments varied the period of preincubation with insulin (10 nM; Fig. 3Go). The increase in DHAA uptake rate followed a 12-h latency period, suggesting a mechanism involving de novo synthesis of transport or regulatory proteins. This possibility was investigated further using cycloheximide, an inhibitor of protein synthesis. Cycloheximide (10 µM; 12 h) prevented stimulation by insulin (10 nM; 12 h) of DHAA uptake (Fig. 4Go).



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  		Figure 3. Stimulation of DHAA uptake rate requires 12-h preincubation with insulin. UMR-106 cells were maintained in serum-free medium for 24 h and preincubated with vehicle (control) or insulin (10 nM) for the indicated periods. Subsequently, the cells were incubated with 10 µM [14C]DHAA for 1 min at 23 C and then harvested. Plotted are the mean ± SEM values from three independent experiments.

 


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  		Figure 4. Cycloheximide prevents insulin stimulation of DHAA uptake. UMR-106 cells were maintained in serum-free medium for 24 h and treated with vehicle (control) or insulin (10 nM) for the final 12 h. Cycloheximide (10 µM) was added at the same time as insulin where indicated. Subsequently, the cells were incubated with 10 µM [14C]DHAA for 1 min at 23 C and then harvested. Plotted are the mean ± SEM from four independent experiments. *, P < 0.05 for effect of insulin.

 
To investigate the nature of the receptor mediating the effect of insulin on DHAA transport, we examined the dependence of the response on insulin and IGF-I concentrations (Fig. 5Go). Half-maximal stimulation of DHAA uptake required 100 pM insulin (Fig. 5Go, top panel). IGF-I also increased the DHAA transport rate, but was less potent than insulin (Fig. 5Go, bottom panel), consistent with involvement of the insulin receptor. Moreover, insulin and IGF-I appeared to be acting through similar mechanisms, because combining 10 nM insulin with 10 nM IGF-I caused the same increase in DHAA transport as that observed with either peptide alone. Rates of uptake of DHAA (10 µM; 23 C), expressed as nanomoles per g cell protein/min, were: vehicle, 57 ± 10; insulin, 122 ± 18; IGF-I, 138 ± 18; and insulin plus IGF-I, 113 ± 18 (n = 3).



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  		Figure 5. Half-maximal stimulation of DHAA uptake rate requires 100 pM insulin. UMR-106 cells were pretreated for 24 h with the indicated concentrations of insulin (top panel) or IGF-I (bottom panel) in serum-free medium. Subsequently, the cells were incubated with 10 µM [14C]DHAA for 1 min at 23 C and then harvested. Plotted are the mean ± SEM values from three independent experiments.

 
Insulin has been reported to increase facilitated hexose transport activity in UMR-106 cells (30, 31). Therefore, we investigated the possibility that DHAA was taken up into osteoblastic cells by facilitative hexose transporters (GLUT). Cells were pretreated with vehicle or insulin (10 nM; 24 h), and the initial rates of uptake of DHAA and 2-deoxyglucose were determined in the presence of cytochalasin B (10 µM; 23 C). DHAA uptake and 2-deoxyglucose uptake were virtually abolished by cytochalasin B, which is a specific inhibitor of facilitative hexose transporters (Fig. 6Go). Furthermore, insulin stimulated the uptake of both DHAA and 2-deoxyglucose.



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  		Figure 6. Cytochalasin B inhibits the uptake of DHAA and 2-deoxyglucose. UMR-106 cells were pretreated for 18–24 h with insulin (10 nM) or vehicle (control) in serum-free medium. Subsequently, the cells were incubated with 10 µM [14C]DHAA 23 C (top panel) or 60 µM [3H]deoxyglucose (bottom panel) for 1 min at 23 C, with or without 10 µM cytochalasin B. Plotted are the mean ± SEM values from four or five independent experiments. *, P < 0.05 for effect of insulin. #, P < 0.05 for effect of cytochalasin B.

 
Next, we tested the acute effects of various unlabeled sugars (10 mM) on the uptake of radiolabeled DHAA and 2-deoxyglucose. Transport of [14C]DHAA was inhibited by D-glucose and 2-deoxyglucose, both of which are substrates of GLUT (Fig. 7Go, top panel). In contrast, uptake was not inhibited by L-glucose or fructose. A similar specificity was seen for inhibition by sugars of 2-[3H]deoxyglucose uptake (Fig. 7Go, bottom panel). We studied the potency with which D-glucose inhibited uptake of [14C]DHAA. Half-maximal inhibition of [14C]DHAA uptake required 3 mM D-glucose (Fig. 8Go). Taken together, these findings indicate that uptake of DHAA is mediated by facilitative hexose transporters.



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  		Figure 7. Uptake of DHAA and 2-deoxyglucose is inhibited by glucose analogs, which are substrates for GLUT. UMR-106 cells were pretreated for 20–24 h with insulin (10 nM) in serum-free medium. Subsequently, the cells were incubated with 10 µM [14C]DHAA (top panel) or 60 µM [3H]deoxyglucose (bottom panel) for 1 min at 23 C, with or without 10 mM of the indicated sugars. Plotted are the mean ± SEM from three to five independent experiments. *, P < 0.05 compared with the glucose-free vehicle treatment.

 


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  		Figure 8. Half-maximal inhibition of DHAA uptake requires 3 mM D-glucose. UMR-106 cells were pretreated for 18 h with insulin (10 nM). Subsequently, the cells were incubated for 1 min at 23 C with 10 µM [14C]DHAA and the indicated concentrations of D-glucose. Plotted are the mean ± SEM from three independent experiments.

 
In contrast to its stimulatory effect on uptake of DHAA, insulin (10 nM; 24 h) had no effect on uptake of ascorbate (reduced vitamin C). We have previously shown that ascorbate is taken up in UMR-106 cells through a specific sodium-ascorbate cotransporter (25). Cotransport activity was assessed by measuring the initial rate of [14C]ascorbate uptake in the presence and absence of DIDS (3 mM), an inhibitor of sodium-ascorbate cotransport (34). Specific uptake was calculated as the difference between total and DIDS-insensitive uptakes. Rates of specific uptake of ascorbate (10 µM; 37 C), expressed as nanomoles per g cell protein/min, were: vehicle, 20 ± 3; and insulin, 22 ± 5 (n = 5).

Stimulation of DHAA uptake and facilitated hexose transport activity by insulin and IGF-I was not accompanied by marked changes in cell morphology, as judged by phase contrast microscopy (not shown). Insulin and IGF-I increased cell protein content only slightly. For example, cell protein contents after 24-h incubation (vitamin C added only for the final minute), expressed as micrograms per culture, were: vehicle, 1088 ± 57 (n = 25); insulin (10 nM), 1245 ± 71 (n = 25); and IGF-I (10 nM), 1374 ± 133 (n = 6).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ascorbate is an enzyme cofactor and antioxidant that stimulates the transcription, translation, and posttranslational processing of collagen in connective tissue cells (35). In cultures of bone-derived cells, ascorbate stimulates osteoblastic differentiation, synthesis, and deposition of collagen as well as mineralization (5, 22, 23). Our previous studies established the existence of specific L-ascorbate transporters in the plasma membrane of calvarial cells and osteoblastic cell lines (5, 25, 26, 34, 36, 37). Kinetic studies revealed a Na+-ascorbate cotransport system that was sensitive to a number of anion transport inhibitors, including DIDS. Ascorbate transport was entirely distinct from hexose transport because, firstly, ascorbate uptake was not altered acutely by the presence of D-glucose or cytochalasin B, and secondly, the rate of ascorbate uptake was significantly lowered in cells pretreated with vitamin C, whereas 2-deoxyglucose uptake was not affected. The present experiments show that osteoblasts can take up and reduce DHAA. Accumulation of intracellular ascorbate from extracellular DHAA has also been reported for human erythrocytes, which lack the Na+-ascorbate cotransporter (38). However, the presence of DHAA uptake and reduction activities in human erythrocytes and erythrocyte ghosts results in a steady state intracellular ascorbate concentration that is no greater than the extracellular ascorbate concentration (25–100 µM) (38, 39), whereas osteoblasts incubated with DHAA can achieve millimolar intracellular concentrations of ascorbate.

Bone growth and remodeling are decreased in insulin-dependent (type I) diabetes mellitus, leading to osteopenia and osteoporosis (6, 7). Conversely, patients with hyperinsulinemia have increased bone mineral density (8). Immunohistochemical staining of neonatal rat calvaria for the insulin receptor showed strong staining in active osteoblasts, in contrast to little staining in periosteal tissues or osteocytes (28). We chose to study the effects of insulin in UMR-106 cells because they express high affinity insulin receptors (27) that regulate Na+-dependent phosphate transport (29) and glucose uptake mediated by facilitative hexose transporters (GLUT) (30, 31). Our results indicate that the vitamin C transport system, which is stimulated by insulin in osteoblastic cells, is selective for the neutral molecule, DHAA, over the anion, ascorbate.

DHAA has been shown to be a transported by GLUT1 expressed in Xenopus oocytes (40). DHAA uptake also appears to be mediated by facilitative hexose transporters in osteoblastic cells, as it is inhibited by cytochalasin B and by glucose analogs that are substrates for GLUT. Further evidence that facilitative hexose transporters mediate DHAA uptake is that half-maximal inhibition of DHAA uptake rate requires 3 mM D-glucose, which corresponds to the apparent Km of GLUT1 for glucose (41).

A latency period of 12 h is required for insulin to markedly increase the DHAA uptake rate in UMR-106 cells, and cycloheximide prevents the increase, consistent with mediation of stimulated DHAA uptake by newly expressed GLUT. UMR-106 cells lack the facilitative hexose transporter isoform (GLUT4) associated with rapid stimulation by insulin (42). Instead, the maximal effect of insulin (10 nM) on facilitated hexose transport activity requires approximately 16 h and is associated with increased expression of GLUT1 and GLUT3 (42). Insulin and IGF-I appear to act though high affinity insulin receptors because of the greater potency of insulin over IGF-I and the absence of additive effects of insulin and IGF-I on DHAA transport.

Under physiological conditions in vivo, virtually all plasma vitamin C is in the form of ascorbate; therefore, accumulation of vitamin C by osteoblasts probably occurs via Na+-ascorbate cotransport (36). However, under pathological conditions, such as wound healing (43) or inflammation (44), extracellular ascorbate is oxidized to DHAA, which may be taken up by cells and reduced to ascorbate. For example, activation of neutrophils results in the production of reactive oxygen species that oxidize extracellular ascorbate to DHAA. DHAA is then taken up by these neutrophils, resulting in rapid elevation of intracellular ascorbate (44). It is possible that a similar mechanism occurs during osseous remodeling and contributes to the coupling of bone formation to osteoclastic bone resorption. Resorbing osteoclasts, which are sources of reactive oxygen species (45, 46), may oxidize extracellular ascorbate. The resulting DHAA may then be taken up and reduced by neighboring preosteoblasts and osteoblasts, stimulating their differentiation and the production of osteoid.

Insulin-dependent (type I) diabetes mellitus may be a state of persistent oxidative stress (17). Ascorbate acts as an antioxidant in compartments where it is sufficiently concentrated. Many studies have shown that people with diabetes have lower plasma concentrations of ascorbate than those without diabetes (14, 15, 16). Therefore, antioxidant defense may be impaired in diabetics. Interestingly, prolonged treatment with ascorbate has been reported to normalize capillary fragility in insulin-dependent diabetics with microangiopathy (47).

Deficient recycling of DHAA to ascorbate under conditions of insulin-dependent diabetes may have pathological effects. DHAA is directly cytotoxic (18, 19, 20). Moreover, injection of DHAA into rats leads to lasting hyperglycemia and death unless insulin is administered (21). The plasma DHAA concentration is negligible in the blood of healthy people, but is persistently elevated in some diabetic patients (14, 16).

We conclude that insulin and IGF-I stimulate the osteoblastic uptake of DHAA through facilitative hexose transporters. The high potency of insulin indicates mediation by insulin receptors. DHAA is reduced intracellularly to ascorbate. Recycling of vitamin C detoxifies the extracellular fluid and maintains high intracellular concentrations of ascorbate. In insulin-dependent (type I) diabetics, DHAA may remain in the extracellular fluid due to decreased transport activity and competitive inhibition by elevated concentrations of glucose. Diminished vitamin C recycling may contribute to the impaired osteoblast function and osteopenia associated with diabetes.


   Acknowledgments
 
We thank E. Pruski for excellent technical assistance.


   Footnotes
 
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Medical Research Council of Canada, and a Medical Research Council development grant (to S.J.D.). Back

Received June 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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