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Histomorphometric analysis of diabetic osteopenia in streptozotocin-induced diabetic mice: A possible role of oxidative stress
Bone 40 (2007) 1408 – 1414 www.elsevier.com/locate/bone
Histomorphometric analysis of diabetic osteopenia in streptozotocin-induced diabetic mice: A possible role of oxidative stress Yasuhiro Hamada a,b , Sohei Kitazawa c , Riko Kitazawa c , Hideki Fujii a , Masato Kasuga b , Masafumi Fukagawa a,b,⁎ b
a Division of Nephrology and Dialysis Center, Kobe University Graduate School of Medicine, Kobe, Japan Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan c Division of Molecular Pathology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, Kobe, Japan
Received 7 September 2006; revised 25 November 2006; accepted 14 December 2006 Available online 24 December 2006
Abstract Diabetic osteopenia causes an increase in bone fracture and a delay in healing of fractures, and affects the quality of life. However, the mechanisms responsible for the disease have not been clearly identified. Oxidative stress may be a potential candidate for the pathogenesis, since it is increased under diabetic conditions and is known to induce cellular dysfunction in a wide variety of cell types. Although in vitro studies have shown that oxidative stress inhibits osteoblastic differentiation and induces osteoblast insults and apoptosis, the relationship between diabetic osteopenia and oxidative stress remains unclear. To explore these issues, analysis of a mouse model that represents the diabetic osteopenia as seen in patients with diabetes is necessary. However, there are few reports of such a model. Therefore, we focused on the streptozotocin (STZ)-induced diabetic mouse, one of the most common animal models of type 1 diabetes. Eight-week-old male C57BL/6 mice were randomly assigned to the following three groups: 1) control group, 2) diabetic group, and 3) insulin-treated diabetic group. After 12 weeks of STZ treatment, the physical properties of the femora, and the static and dynamic parameters of bone histomorphometry of the tibiae from STZ-induced diabetic mice (STZmice) were assessed, and oxidative stress in the whole body and bone of the mice was evaluated. Renal function was comparable in all three groups at the end of the experimental period. In addition, no significant difference in serum PTH, Ca, and P was found among the three groups. In contrast, radiological analysis demonstrated a significant decrease in trabecular bone volume, and histomorphometric analyses confirmed that parameters for both bone formation (OV/BV, OS/BS, and BFR/BS) and bone resorption (ES/BS and Oc.S/BS) were also significantly lower in STZ-mice. In addition, urinary excretion of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, was elevated in STZ-mice. Further immunohistological studies showed intensified immunostaining of an oxidative stress marker in bone tissue including the osteoblasts of diabetic mice. Here, we demonstrated that STZ-mice exhibit low-turnover osteopenia associated with increased oxidative stress. © 2006 Elsevier Inc. All rights reserved. Keywords: Diabetic osteopenia; Low-turnover bone; Osteoblast; Oxidative stress; Streptozotocin
Introduction Diabetes mellitus causes many complications such as nephropathy, neuropathy, and retinopathy. Osteopenia is also a complication of diabetes [1–3]. Diabetic osteopenia causes an increase in bone fracture [4,5] and a delay in healing of fractures [6,7], and affects the quality of life. However, there are few ⁎ Corresponding author. Division of Nephrology and Dialysis Center, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Fax: +81 78 382 6509. E-mail address: [email protected] (M. Fukagawa). 8756-3282/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2006.12.057
optimal therapies for the disorder. Furthermore, although many human and experimental studies on the complications of diabetes mellitus have demonstrated extensive alterations in bone and mineral metabolism [8,9], the mechanisms responsible for diabetic osteopenia have not been clearly identified. Many derangements associated with diabetes, such as oxidative stress, hyperglycemia, and body weight loss, can be considered to be involved in the pathogenesis of diabetic osteopenia. Among them, oxidative stress may be a potential candidate, since it is known to induce cellular dysfunction in a wide variety of cell types. Oxidative stress is induced by a variety of mechanisms including the glycation reaction [10], the
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Table 1 Metabolic measurements
Serum Cr (mg/dl) PTH (pg/ml) Serum Ca (mg/dl) Serum P (mg/dl) Serum Glucose (mmol/L)
Fig. 1. Time course of changes in body weight (A) and HbA1c (B). Data are mean ± S.D. Open circle = control group; closed circle = diabetic group; open square = insulin-treated diabetic group. *p < 0.01 vs. control group.
polyol pathway [11], protein kinase C-dependent activation of membranous NADPH oxidase [12], and the mitochondrial electron transport chain [13,14] under diabetic conditions, and has been suggested to play an important role in the pathogenesis of diabetic microangiopathies such as nephropathy, neuropathy, and retinopathy. Concerning bone metabolism, in vitro studies have shown that oxidative stress inhibits osteoblastic differentiation [15,16] and induces osteoblast insults and apoptosis [17,18]. Thus, oxidative stress may be related to the pathogenesis of diabetic osteopenia. To investigate the mechanisms by which diabetes affects bone and explore the relationship between oxidative stress and diabetic osteopenia, an animal model that represents the diabetic osteopenia as seen in patients with diabetes is necessary. We focused on the streptozotocin (STZ)-induced diabetic mouse, which is one of the most common animal models of type 1 diabetes. There are many reports on diabetic microangiopathy in the mouse. However, there are few detailed reports on osteopenia in STZ-induced diabetic mice. In the current study, we assessed the physical properties of the femora, and the static and dynamic parameters of bone histomorphometry of the tibiae from STZ-induced diabetic mice, and evaluated oxidative stress in the whole body and bone of the mice.
Control
DM
Insulin
0.36 ± 0.07 42.5 ± 7.8 8.7 ± 0.5 8.2 ± 1.3 6.81 ± 1.34
0.38 ± 0.09 46.1 ± 17.9 7.9 ± 1.2 9.7 ± 1.4 34.53 ± 14.83*
0.35 ± 0.05 37.4 ± 20.1 9.0 ± 0.9 8.8 ± 1.3 7.67 ± 1.65
Data were obtained after 12 weeks of STZ or citrate vehicle injection. Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Data are means ± S.D. *p < 0.01 vs. control group. levels were measured at 0, 4, and 12 weeks after STZ or vehicle injection. HbA1c levels were determined using a DCA2000 analyzer (Bayer Medical, Tokyo, Japan) using venous blood from the tail.
Serum measurements The mice were killed under ether anesthesia to obtain blood via cardiac puncture. Blood serum was prepared from each sample by centrifugation for 5 min at 3000 rpm. Serum samples were stored at −80 °C until analysis. Serum creatinine levels were measured using Spotchem EZ SP 4430 (Arkray Inc., Kyoto, Japan) according to the manufacturer's instructions. Serum concentrations of parathyroid hormone (PTH) were determined using a mouse PTH ELISA kit (Immutopics Inc., San Clemente, CA, USA). Serum concentrations of Ca and P were determined by the Calcium-E test and the Phospha-C test (both from Wako, Osaka, Japan), respectively.
Sample preparation and skeletal morphology Bone radiographs of excised femora were taken with a soft X-ray apparatus (Type SRO-M50; Sofron, Tokyo, Japan). The BMDs of the left femora were measured by single energy X-ray absorptiometry using a bone mineral analyzer
Materials and methods Animals and experimental design Eight-week-old male C57BL/6 mice weighing 19–26 g were randomly assigned to control, diabetic, or insulin-treated diabetic groups. The number of animals per experimental group was eight. Mice in the diabetic and insulintreated diabetic groups were intraperitoneally injected with STZ (Sigma Chemical Co., St Louis, MO, USA) (100 mg/kg body weight in 100 μL of sterile citrate buffer, pH 4.5) on two consecutive days. Control group mice were injected with citrate vehicle alone. Mice with venous blood glucose levels of over 17 mmol/L, in samples obtained from the tail and measured by Glutest-Ace (Sanwa Kagaku Kenkyusho, Nagoya, Japan), were considered diabetic. Insulin treatment was initiated when blood glucose levels reached more than 17 mmol/L in the insulin-treated diabetic group. The mice were treated with Linbit insulin implants (LinShin Canada Inc., Scarborough, Ontario, Canada), which release a controlled amount of insulin. All mice had free access to standard chow and tap water throughout the experiment. The procedures were approved by the Institutional Animal Care and Use Committee guidelines of Kobe University Graduate School of Medicine. In preliminary experiments, there was no significant difference in bone mineral density (BMD) among the three groups at 4 weeks after STZ injection (data not shown). Therefore, the mice were followed for 12 weeks after STZ injection. Body weight and hemoglobin A1c (HbA1c)
Fig. 2. Bone X-ray and bone mineral density. The femora were removed after 12 weeks of STZ or vehicle treatment. Diabetic mice showed severe osteopenia in femora by X-ray (A). The femora were analyzed by dividing them longitudinally into 3 equal regions, and BMD of each fraction was measured (B). Data are mean ± S.D. Open circle = control group; closed circle = diabetic group; open square = insulin-treated diabetic group. *p < 0.01 vs. control group.
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Fig. 3. Villanueva–Goldner stain and bone formation parameters. The tibiae were removed after 12 weeks of STZ or vehicle treatment. Compared with control mice (A) and insulin-treated diabetic mice (C), decreases of trabecular bones stained green and osteoid volume stained red in diabetic mice (B) were confirmed. All bone formation parameters were significantly decreased in diabetic group compared with other two groups (D–F). Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Magnification is 100×. Data are means ± S.D. *p < 0.05 vs. control group. (DCS-600R; Aloka Co., Tokyo, Japan). All histological analyses were carried out using mice at 12 weeks after STZ treatment. For Villanueva–Goldner staining, the right tibiae were excised, fixed with 70% ethanol, embedded in methyl methacrylate, and sectioned in 6-μm slices. For double labeling, mice were injected subcutaneously with calcein (8 mg/kg body weight) at 10 days and 3 days before sacrifice. Tartrate-resistant acid phosphatase (TRAP)-positive cells were stained at pH 5.0 in the presence of L(+)-tartaric acid using naphthol AS-MX phosphate (Sigma Chemical Co., St. Louis, MO, USA) in N,Ndimethyl formamide as the substrate. The specimens were subjected to histomorphometric analysis under a light microscope with a micrometer, using an image analyzer (System Supply Co., Nagano, Japan). Parameters for the trabecular bone were measured in an area of 1.2 mm in length from 0.1 mm below the growth plate at the proximal metaphysis of the tibiae. All parameters comply with the guidelines of the nomenclature committee of the American Society of Bone and Mineral Research [19].
Assessment of systemic oxidative stress To evaluate the extent of oxidative stress in the animals, urinary 8hydroxydeoxyguanosine (8-OHdG), a sensitive indicator of oxidative DNA
damage, was measured. After 12 weeks of STZ treatment, a 24 h-urine collection from each mouse using metabolic cages was conducted. The urine was kept at −20 °C until the concentrations of 8-OHdG were measured. Urinary 8-OHdG was determined using a sandwich enzyme-linked immunosorbent assay kit (NOF Corporation, Tokyo, Japan).
Immunohistochemistry The right femora for immunohistochemistry were excised, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2) for 3 days at room temperature, and decalcified with 20% EDTA in 0.1 M PB at room temperature for 4 days. The tissue samples were then dehydrated through a graded ethanol series, embedded in paraffin, and sectioned in 6-μm slices. Formation of 8-OHdG in bone tissues was assessed with anti-8-OHdG mouse monoclonal antibodies (NOF Corporation, Tokyo, Japan). In brief, the slices were pre-incubated with blocking agent (Simple Stain mouse system; Nichirei, Tokyo, Japan), followed by incubation with the primary antibodies described above for 60 min at room temperature. A universal immuno-peroxidase polymer (Histofine™ Simple Stain MAX PO system, anti-mouse and rabbit; Nichirei, Tokyo, Japan) was used for immunostaining. The percentage of 8-
Fig. 4. Mineral apposition rate. The tibiae were removed after 12 weeks of STZ or vehicle treatment. Compared with control mice (A) and insulin-treated diabetic mice (C), decreases of calcein double labeling in diabetic mice (B) were confirmed. MAR (D), dLS/BS (E), and BFR/BS (F) were significantly decreased in diabetic group compared with other two groups. Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Magnification is 100×. Data are means ± S.D. *p < 0.05 vs. control group.
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OHdG-positive cells in bone tissue was determined by counting the cells in a 2 × 2 mm area of metaphyseal bone marrow tissue. All evaluations were performed in a blinded fashion.
Table 2 Assessment of oxidative stress
Statistical analysis
Urinary 8-OHdG (ng/day) Percentage of positive cells (%)
Results were expressed as mean ± S.D. Data were examined by one-way ANOVA followed by Tukey's HSD test. Associations between the markers of oxidative stress and the histomorphometric parameters of bone formation were determined using Spearman rank correlation analysis. All statistical analyses were performed using SPSS for Windows ver. 12.0 (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.
Data were obtained after 12 weeks of STZ or citrate vehicle injection. Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Data are means ± S.D. *p < 0.01 vs. control group. †p < 0.05 vs. control group.
Control
DM
Insulin
0.58 ± 0.45 11.6 ± 2.8
65.64 ± 29.21* 25.2 ± 6.7†
1.03 ± 1.04 12.6 ± 5.9
Results
groups at the end of the experimental period. Furthermore, no significant differences in serum PTH, Ca, and P were found among the groups (Table 1).
Animal profiles
Bone X-ray and bone mineral density
Changes in body weight and HbA1c levels during the study are shown in Fig. 1. Body weight increased steadily in control mice throughout the 12-week observation period. In contrast, diabetic mice showed little gain in weight. Thus, the body weights of the diabetic animals were significantly (p < 0.01) reduced compared with the controls at the end of the experiment (Fig. 1A). HbA1c levels in the control group did not change throughout the experimental period. In contrast, HbA1c levels were significantly (p < 0.01) higher in the diabetic groups than in the control group during the experiment (Fig. 1B). These data indicate that hyperglycemia in diabetic mice was consistent and not transient. However, insulin treatment rescued these changes (Fig. 1).
No significant differences in bone shape could be detected between control, diabetic, and insulin-treated diabetic mice. However, diabetic mice showed severe osteopenia in the femora by X-ray (Fig. 2A). The BMD of the femora was decreased by about 15–20% in diabetic mice after 12 weeks of STZ treatment compared with that in the control mice (Fig. 2B). To assess the distribution of BMD in the femora, the bones were divided longitudinally into three equal parts, and the BMD of each part was measured (Fig. 2B). The BMD of each part was decreased to a similar extent in diabetic mice, suggesting that trabecular bone and cortex bone were equally affected by diabetic condition. Insulin treatment resolved the osteopenia and the reduction of BMD (Fig. 2).
Metabolic measurements
Bone histomorphometry
The metabolic measurements made at the end of the 12-week experimental period are summarized in Table 1. These data demonstrate that renal function was comparable in all three
Villanueva–Goldner staining indicated decreases of trabecular bone, stained green, and osteoid volume, stained red, in diabetic mice compared with control mice and insulin-treated
Fig. 5. TRAP stain and bone resorption parameters. The tibiae were removed after 12 weeks of STZ or vehicle treatment. The number of TRAP positive osteoclasts was lower in diabetic mice (B) than in control mice (A) and insulin-treated diabetic mice (C). Both bone resorption parameters were significantly decreased in diabetic group compared with other two groups (D, E). Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Magnification is 400×. Data are means ± S.D. *p < 0.05 vs. control group.
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diabetic mice (Figs. 3A–C). Histomorphometric measurements revealed that bone formation parameters [osteoid volume/ bone volume (OV/BV), osteoid surface/bone surface (OS/BS), and osteoid thickness (O.Th)] were significantly decreased in the diabetic group compared with the other two groups (Figs. 3D–F). Calcein double labeling showed that the width between the two stained labels was narrower in diabetic mice than in control mice and insulin-treated diabetic mice (Figs. 4A–C). The mineral apposition rate (MAR), reflecting the ability of individual osteoblasts to form bone, was about 50% decreased and consequently, the bone formation rate (BFR/BS), determined by the number and function of osteoblasts, was significantly lower in diabetic mice than in control mice and insulin-treated diabetic mice (Figs. 4D–F). Histological analysis of the proximal tibiae revealed that the number of TRAP-positive osteoclasts was lower in diabetic mice than in control mice and insulin-treated diabetic mice (Figs. 5A–C). Histomorphometric measurements supported the observation that bone resorption parameters [eroded surface (ES/BS) and osteoclast surface (Oc.S/BS)] were significantly lower in diabetic mice (Figs. 5D and E). The parameters for both bone formation and bone resorption were also significantly lower in diabetic mice, indicating that STZ-induced diabetic mice suppressed both bone formation and resorption, which is similar to that seen in humans.
Table 3 Correlations between the markers of oxidative stress and the histomorphometric parameters of bone formation OV/BV
OS/BS
O.Th
MAR
BFR/BS
r
r
r
r
r
Urinary 8-OHdG (ng/day) −0.721* − 0.864* − 0.447† − 0.815* − 0.893* Percentage of positive cells −0.725* − 0.877* − 0.464† − 0.818* − 0.94* (%) r is Spearman correlation coefficients. Data of control group, diabetic group, and insulin-treated diabetic group are combined for the analysis. *p value <0.01. †p value <0.05.
To assess the oxidative stress in bone tissues, immunohistochemical staining for 8-OHdG (Fig. 6) was performed. Staining of 8-OHdG was clearly intensified in the bone tissues, including osteoblasts, of diabetic mice (Figs. 6B, E) compared with control mice (Figs. 6A, D). In contrast, staining was prominently attenuated in insulin-treated diabetic mice (Figs. 6C, F). Moreover, the intensity of the staining in insulin-treated diabetic mice was similar to that in the control mice. Quantitative analysis confirmed these results (Table 2). Taken together, these results demonstrate that oxidative stress is increased not only in the whole body, but also in bone tissues, including the osteoblasts of diabetic animals. Moreover, the results demonstrate that insulin treatment rescues both lowturnover osteopenia and increased oxidative stress.
Assessment of oxidative stress Associations between oxidative stress and bone formation Urinary 8-OHdG, a sensitive indicator of oxidative DNA damage, was markedly (p < 0.01) higher in diabetic mice than in control mice. However, insulin-treated diabetic mice showed suppressed excretion of the marker, resulting in an absence of any significant difference with the control mice (Table 2).
Table 3 shows Spearman correlations of the markers of oxidative stress with the histomorphometric parameters of bone formation in all three groups combined. The markers of oxidative stress (urinary excretion of 8-OHdG and percentage
Fig. 6. Assessment of oxidative stress. Immunohistochemical staining for 8-OHdG revealed that staining to 8-OHdG was clearly intensified in bone tissues (B) including osteoblasts (E, arrowhead) of diabetic mice compared with control mice (A, D) and insulin-treated diabetic mice (C, F). A–C: Magnification is 200×. D–F: Magnification is 400×.
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of 8-OHdG) were inversely associated with the histomorphometric parameters of bone formation (OV/BV, OS/BS, O.Th, MAR, and BFR/BS). Discussion In the current study, our radiological analysis demonstrated a significant decrease in mouse trabecular bone volume (by about 15–20%) 12 weeks after the induction of diabetes, and our histological analysis confirmed that parameters for both bone formation (OV/BV, OS/BS, and BFR/BS) and resorption (ES/ BS and Oc.S/BS) were also significantly lower in STZ-induced diabetic mice. These results indicate that both bone formation and resorption are suppressed in STZ-induced diabetic mice, which simulate diabetic osteopenia. To exclude potential secondary effects of STZ treatment, so-called STZ toxicity, we also examined an insulin-treated diabetic group. Insulin treatment largely restored reduced BMD and prevented the suppression of both bone formation and resorption observed in the diabetic group, indicating that the osteopenia in STZinduced diabetic mice was strictly related to the diabetic state. In the present study, we selected the STZ-induced diabetic mouse from various animal models to characterize the phenotype of bone in diabetes. Although studies have examined the influence of diabetes on rat bone [20–22], few have directly addressed the influence of diabetes on the skeletal system of mice, an animal model that is acceptable for genetic manipulation. Consistent with rat studies [20–22] and human observations [1–3], we found a decrease in bone volume in the mouse model. Moreover, we demonstrated that the osteopenia in STZinduced diabetic mice exhibited low bone turnover. In the near future, these mice may be an adequate animal model to clarify the mechanisms responsible for low-turnover osteopenia as well as diabetic osteopenia through knockout and/or transgenic approaches. In vitro studies have reported that high glucose influences osteoblast differentiation [23], impairs bone formation [24], and inhibits bone mineralization [25]. In addition, experimental and clinical studies have demonstrated that osteoblast function is impaired under diabetic conditions [26,27]. Consistent with these reports, bone histomorphometry in the present study confirmed that osteoblast function and mineralization were impaired in STZ-induced diabetic osteopenia. Taken together, our results suggest that diabetic osteopenia without renal dysfunction is mainly caused by inhibition of osteoblast formation and function. Concerning the pathogenesis of osteoblast dysfunction, oxidative stress may be an important factor. In the present study, urinary excretion of 8-OHdG, a marker of oxidative DNA damage, was elevated in STZ-induced diabetic mice, while the marker was significantly suppressed to almost control levels in insulin-treated diabetic mice. Further immunohistological studies showed intensified immunostaining of the oxidative stress marker in bone tissue, including the osteoblasts, of diabetic mice, which was again diminished in insulin-treated diabetic mice. These findings indicate that oxidative stress in diabetic conditions was suppressed in insulin-treated diabetic
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mice accompanied by recovery from diabetic osteopenia. In vitro studies have shown that oxidative stress inhibits osteoblastic differentiation [15,16] and induces osteoblast insults and apoptosis [17,18]. However, there are few reports on the in vivo relationship between oxidative stress and diabetic osteopenia. The observations in the present study directly identify increased oxidative stress in diabetic bone as well as the whole body. In addition, we demonstrated that the markers of oxidative stress (urinary excretion of 8-OHdG and percentage of 8-OHdG positive cells) were inversely associated with the histomorphometric parameters of bone formation (OV/BV, OS/BS, O.Th, MAR, and BFR/BS). Although the present study did not fully clarify the main cause of diabetic osteopenia, our results suggest that the increase in oxidative stress may at least partly contribute to the development of diabetic osteopenia. To clarify the mechanism of the disease, further studies linking diabetic osteopenia to oxidative stress and bone turnover are planned. In conclusion, we demonstrated here that STZ-induced diabetic mice suppressed both bone formation and resorption associated with increased oxidative stress. These findings suggest that STZ-induced diabetic mice can be a useful animal model for diabetic osteopenia as seen in patients with diabetes. Furthermore, oxidative stress may be a therapeutic target in diabetic osteopenia. Acknowledgments This work is supported by the 21st Century COE Program “Center of Excellence for Signal Transduction Disease: Diabetes Mellitus as Model” awarded to M.K. by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We are grateful to the hard tissue research team at Kureha Chemical Co. for technical assistance. We also thank T. Nii-Kono and S. Matsuda (Kobe University Graduate School of Medicine) for their technical assistance. References [1] Kayath MJ, Dib SA, Vieira JG. Prevalence and magnitude of osteopenia associated with insulin-dependent diabetes mellitus. J Diabetes Complications 1994;8:97–104. [2] Kemink SA, Hermus AR, Swinkels LM, Lutterman JA, Smals AG. Osteopenia in insulin-dependent diabetes mellitus; prevalence and aspects of pathophysiology. J Endocrinol Invest 2000;23:295–303. [3] Lopez-Ibarra PJ, Pastor MM, Escobar-Jimenez F, Pardo MD, Gonzalez AG, Luna JD, et al. Bone mineral density at time of clinical diagnosis of adult-onset type 1 diabetes mellitus. Endocr Pract 2001 (Sep–Oct);7 (5):346–51. [4] Bouillon R. Diabetic bone disease. Calcif Tissue Int 1991;49:155–60. [5] Forsen L, Meyer HE, Midthjell K, Edna TH. Diabetes mellitus and the incidence of hip fracture: results from the Nord-Trondelag Health Survey. Diabetologia 1999;42:920–5. [6] Cozen L. Does diabetes delay fracture healing? Clin Orthop Relat Res 1972;82:134–40. [7] Herskind AM, Christensen K, Norgaard-Andersen K, Andersen JF. Diabetes mellitus and healing of closed fractures. Diabetes Metab 1992;18: 63–64. [8] Levin ME, Boisseau VC, Avioli LV. Effects of diabetes mellitus on bone mass in juvenile and adult-onset diabetes. N Engl J Med 1976;294:241–5. [9] Seino Y, Ishida H. Diabetic osteopenia: pathophysiology and clinical aspects. Diabetes Metab Rev 1995;11:21–35.
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[19] Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987;2:595–610. [20] Sasaki T, Kaneko H, Ramamurthy NS, Golub LM. Tetracycline administration restores osteoblast structure and function during experimental diabetes. Anat Rec 1991;231:25–34. [21] Spanheimer RG, Umpierrez GE, Stumpf V. Decreased collagen production in diabetic rats. Diabetes 1988;37:371–6. [22] Weiss RE, Gorn AH, Nimni ME. Abnormalities in the biosynthesis of cartilage and bone proteoglycans in experimental diabetes. Diabetes 1981;30:670–7. [23] Zayzafoon M, Stell C, Irwin R, McCabe LR. Extracellular glucose influences osteoblast differentiation and c-Jun expression. J Cell Biochem 2000;79:301–10. [24] Terada M, Inaba M, Yano Y, Hasuma T, Nishizawa Y, Morii H, et al. Growth-inhibitory effect of a high glucose concentration on osteoblast-like cells. Bone 1998;22:17–23. [25] Balint E, Szabo P, Marshall CF, Sprague SM. Glucose-induced inhibition of in vitro bone mineralization. Bone 2001;28:21–8. [26] Thrailkill KM, Liu L, Wahl EC, Bunn RC, Perrien DS, Cockrell GE, et al. Bone formation is impaired in a model of type 1 diabetes. Diabetes 2005;54: 2875–2881. [27] Bouillon R, Bex M, Van Herck E, Laureys J, Dooms L, Lesaffre E, et al. Influence of age, sex, and insulin on osteoblast function: osteoblast dysfunction in diabetes mellitus. J Clin Endocrinol Metab 1995; 80:1194–202.
Histomorphometric analysis of diabetic osteopenia in streptozotocin-induced diabetic mice: A possible role of oxidative stress Yasuhiro Hamada a,b , Sohei Kitazawa c , Riko Kitazawa c , Hideki Fujii a , Masato Kasuga b , Masafumi Fukagawa a,b,⁎ b
a Division of Nephrology and Dialysis Center, Kobe University Graduate School of Medicine, Kobe, Japan Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan c Division of Molecular Pathology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, Kobe, Japan
Received 7 September 2006; revised 25 November 2006; accepted 14 December 2006 Available online 24 December 2006
Abstract Diabetic osteopenia causes an increase in bone fracture and a delay in healing of fractures, and affects the quality of life. However, the mechanisms responsible for the disease have not been clearly identified. Oxidative stress may be a potential candidate for the pathogenesis, since it is increased under diabetic conditions and is known to induce cellular dysfunction in a wide variety of cell types. Although in vitro studies have shown that oxidative stress inhibits osteoblastic differentiation and induces osteoblast insults and apoptosis, the relationship between diabetic osteopenia and oxidative stress remains unclear. To explore these issues, analysis of a mouse model that represents the diabetic osteopenia as seen in patients with diabetes is necessary. However, there are few reports of such a model. Therefore, we focused on the streptozotocin (STZ)-induced diabetic mouse, one of the most common animal models of type 1 diabetes. Eight-week-old male C57BL/6 mice were randomly assigned to the following three groups: 1) control group, 2) diabetic group, and 3) insulin-treated diabetic group. After 12 weeks of STZ treatment, the physical properties of the femora, and the static and dynamic parameters of bone histomorphometry of the tibiae from STZ-induced diabetic mice (STZmice) were assessed, and oxidative stress in the whole body and bone of the mice was evaluated. Renal function was comparable in all three groups at the end of the experimental period. In addition, no significant difference in serum PTH, Ca, and P was found among the three groups. In contrast, radiological analysis demonstrated a significant decrease in trabecular bone volume, and histomorphometric analyses confirmed that parameters for both bone formation (OV/BV, OS/BS, and BFR/BS) and bone resorption (ES/BS and Oc.S/BS) were also significantly lower in STZ-mice. In addition, urinary excretion of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, was elevated in STZ-mice. Further immunohistological studies showed intensified immunostaining of an oxidative stress marker in bone tissue including the osteoblasts of diabetic mice. Here, we demonstrated that STZ-mice exhibit low-turnover osteopenia associated with increased oxidative stress. © 2006 Elsevier Inc. All rights reserved. Keywords: Diabetic osteopenia; Low-turnover bone; Osteoblast; Oxidative stress; Streptozotocin
Introduction Diabetes mellitus causes many complications such as nephropathy, neuropathy, and retinopathy. Osteopenia is also a complication of diabetes [1–3]. Diabetic osteopenia causes an increase in bone fracture [4,5] and a delay in healing of fractures [6,7], and affects the quality of life. However, there are few ⁎ Corresponding author. Division of Nephrology and Dialysis Center, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Fax: +81 78 382 6509. E-mail address: [email protected] (M. Fukagawa). 8756-3282/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2006.12.057
optimal therapies for the disorder. Furthermore, although many human and experimental studies on the complications of diabetes mellitus have demonstrated extensive alterations in bone and mineral metabolism [8,9], the mechanisms responsible for diabetic osteopenia have not been clearly identified. Many derangements associated with diabetes, such as oxidative stress, hyperglycemia, and body weight loss, can be considered to be involved in the pathogenesis of diabetic osteopenia. Among them, oxidative stress may be a potential candidate, since it is known to induce cellular dysfunction in a wide variety of cell types. Oxidative stress is induced by a variety of mechanisms including the glycation reaction [10], the
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Table 1 Metabolic measurements
Serum Cr (mg/dl) PTH (pg/ml) Serum Ca (mg/dl) Serum P (mg/dl) Serum Glucose (mmol/L)
Fig. 1. Time course of changes in body weight (A) and HbA1c (B). Data are mean ± S.D. Open circle = control group; closed circle = diabetic group; open square = insulin-treated diabetic group. *p < 0.01 vs. control group.
polyol pathway [11], protein kinase C-dependent activation of membranous NADPH oxidase [12], and the mitochondrial electron transport chain [13,14] under diabetic conditions, and has been suggested to play an important role in the pathogenesis of diabetic microangiopathies such as nephropathy, neuropathy, and retinopathy. Concerning bone metabolism, in vitro studies have shown that oxidative stress inhibits osteoblastic differentiation [15,16] and induces osteoblast insults and apoptosis [17,18]. Thus, oxidative stress may be related to the pathogenesis of diabetic osteopenia. To investigate the mechanisms by which diabetes affects bone and explore the relationship between oxidative stress and diabetic osteopenia, an animal model that represents the diabetic osteopenia as seen in patients with diabetes is necessary. We focused on the streptozotocin (STZ)-induced diabetic mouse, which is one of the most common animal models of type 1 diabetes. There are many reports on diabetic microangiopathy in the mouse. However, there are few detailed reports on osteopenia in STZ-induced diabetic mice. In the current study, we assessed the physical properties of the femora, and the static and dynamic parameters of bone histomorphometry of the tibiae from STZ-induced diabetic mice, and evaluated oxidative stress in the whole body and bone of the mice.
Control
DM
Insulin
0.36 ± 0.07 42.5 ± 7.8 8.7 ± 0.5 8.2 ± 1.3 6.81 ± 1.34
0.38 ± 0.09 46.1 ± 17.9 7.9 ± 1.2 9.7 ± 1.4 34.53 ± 14.83*
0.35 ± 0.05 37.4 ± 20.1 9.0 ± 0.9 8.8 ± 1.3 7.67 ± 1.65
Data were obtained after 12 weeks of STZ or citrate vehicle injection. Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Data are means ± S.D. *p < 0.01 vs. control group. levels were measured at 0, 4, and 12 weeks after STZ or vehicle injection. HbA1c levels were determined using a DCA2000 analyzer (Bayer Medical, Tokyo, Japan) using venous blood from the tail.
Serum measurements The mice were killed under ether anesthesia to obtain blood via cardiac puncture. Blood serum was prepared from each sample by centrifugation for 5 min at 3000 rpm. Serum samples were stored at −80 °C until analysis. Serum creatinine levels were measured using Spotchem EZ SP 4430 (Arkray Inc., Kyoto, Japan) according to the manufacturer's instructions. Serum concentrations of parathyroid hormone (PTH) were determined using a mouse PTH ELISA kit (Immutopics Inc., San Clemente, CA, USA). Serum concentrations of Ca and P were determined by the Calcium-E test and the Phospha-C test (both from Wako, Osaka, Japan), respectively.
Sample preparation and skeletal morphology Bone radiographs of excised femora were taken with a soft X-ray apparatus (Type SRO-M50; Sofron, Tokyo, Japan). The BMDs of the left femora were measured by single energy X-ray absorptiometry using a bone mineral analyzer
Materials and methods Animals and experimental design Eight-week-old male C57BL/6 mice weighing 19–26 g were randomly assigned to control, diabetic, or insulin-treated diabetic groups. The number of animals per experimental group was eight. Mice in the diabetic and insulintreated diabetic groups were intraperitoneally injected with STZ (Sigma Chemical Co., St Louis, MO, USA) (100 mg/kg body weight in 100 μL of sterile citrate buffer, pH 4.5) on two consecutive days. Control group mice were injected with citrate vehicle alone. Mice with venous blood glucose levels of over 17 mmol/L, in samples obtained from the tail and measured by Glutest-Ace (Sanwa Kagaku Kenkyusho, Nagoya, Japan), were considered diabetic. Insulin treatment was initiated when blood glucose levels reached more than 17 mmol/L in the insulin-treated diabetic group. The mice were treated with Linbit insulin implants (LinShin Canada Inc., Scarborough, Ontario, Canada), which release a controlled amount of insulin. All mice had free access to standard chow and tap water throughout the experiment. The procedures were approved by the Institutional Animal Care and Use Committee guidelines of Kobe University Graduate School of Medicine. In preliminary experiments, there was no significant difference in bone mineral density (BMD) among the three groups at 4 weeks after STZ injection (data not shown). Therefore, the mice were followed for 12 weeks after STZ injection. Body weight and hemoglobin A1c (HbA1c)
Fig. 2. Bone X-ray and bone mineral density. The femora were removed after 12 weeks of STZ or vehicle treatment. Diabetic mice showed severe osteopenia in femora by X-ray (A). The femora were analyzed by dividing them longitudinally into 3 equal regions, and BMD of each fraction was measured (B). Data are mean ± S.D. Open circle = control group; closed circle = diabetic group; open square = insulin-treated diabetic group. *p < 0.01 vs. control group.
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Fig. 3. Villanueva–Goldner stain and bone formation parameters. The tibiae were removed after 12 weeks of STZ or vehicle treatment. Compared with control mice (A) and insulin-treated diabetic mice (C), decreases of trabecular bones stained green and osteoid volume stained red in diabetic mice (B) were confirmed. All bone formation parameters were significantly decreased in diabetic group compared with other two groups (D–F). Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Magnification is 100×. Data are means ± S.D. *p < 0.05 vs. control group. (DCS-600R; Aloka Co., Tokyo, Japan). All histological analyses were carried out using mice at 12 weeks after STZ treatment. For Villanueva–Goldner staining, the right tibiae were excised, fixed with 70% ethanol, embedded in methyl methacrylate, and sectioned in 6-μm slices. For double labeling, mice were injected subcutaneously with calcein (8 mg/kg body weight) at 10 days and 3 days before sacrifice. Tartrate-resistant acid phosphatase (TRAP)-positive cells were stained at pH 5.0 in the presence of L(+)-tartaric acid using naphthol AS-MX phosphate (Sigma Chemical Co., St. Louis, MO, USA) in N,Ndimethyl formamide as the substrate. The specimens were subjected to histomorphometric analysis under a light microscope with a micrometer, using an image analyzer (System Supply Co., Nagano, Japan). Parameters for the trabecular bone were measured in an area of 1.2 mm in length from 0.1 mm below the growth plate at the proximal metaphysis of the tibiae. All parameters comply with the guidelines of the nomenclature committee of the American Society of Bone and Mineral Research [19].
Assessment of systemic oxidative stress To evaluate the extent of oxidative stress in the animals, urinary 8hydroxydeoxyguanosine (8-OHdG), a sensitive indicator of oxidative DNA
damage, was measured. After 12 weeks of STZ treatment, a 24 h-urine collection from each mouse using metabolic cages was conducted. The urine was kept at −20 °C until the concentrations of 8-OHdG were measured. Urinary 8-OHdG was determined using a sandwich enzyme-linked immunosorbent assay kit (NOF Corporation, Tokyo, Japan).
Immunohistochemistry The right femora for immunohistochemistry were excised, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2) for 3 days at room temperature, and decalcified with 20% EDTA in 0.1 M PB at room temperature for 4 days. The tissue samples were then dehydrated through a graded ethanol series, embedded in paraffin, and sectioned in 6-μm slices. Formation of 8-OHdG in bone tissues was assessed with anti-8-OHdG mouse monoclonal antibodies (NOF Corporation, Tokyo, Japan). In brief, the slices were pre-incubated with blocking agent (Simple Stain mouse system; Nichirei, Tokyo, Japan), followed by incubation with the primary antibodies described above for 60 min at room temperature. A universal immuno-peroxidase polymer (Histofine™ Simple Stain MAX PO system, anti-mouse and rabbit; Nichirei, Tokyo, Japan) was used for immunostaining. The percentage of 8-
Fig. 4. Mineral apposition rate. The tibiae were removed after 12 weeks of STZ or vehicle treatment. Compared with control mice (A) and insulin-treated diabetic mice (C), decreases of calcein double labeling in diabetic mice (B) were confirmed. MAR (D), dLS/BS (E), and BFR/BS (F) were significantly decreased in diabetic group compared with other two groups. Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Magnification is 100×. Data are means ± S.D. *p < 0.05 vs. control group.
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OHdG-positive cells in bone tissue was determined by counting the cells in a 2 × 2 mm area of metaphyseal bone marrow tissue. All evaluations were performed in a blinded fashion.
Table 2 Assessment of oxidative stress
Statistical analysis
Urinary 8-OHdG (ng/day) Percentage of positive cells (%)
Results were expressed as mean ± S.D. Data were examined by one-way ANOVA followed by Tukey's HSD test. Associations between the markers of oxidative stress and the histomorphometric parameters of bone formation were determined using Spearman rank correlation analysis. All statistical analyses were performed using SPSS for Windows ver. 12.0 (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.
Data were obtained after 12 weeks of STZ or citrate vehicle injection. Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Data are means ± S.D. *p < 0.01 vs. control group. †p < 0.05 vs. control group.
Control
DM
Insulin
0.58 ± 0.45 11.6 ± 2.8
65.64 ± 29.21* 25.2 ± 6.7†
1.03 ± 1.04 12.6 ± 5.9
Results
groups at the end of the experimental period. Furthermore, no significant differences in serum PTH, Ca, and P were found among the groups (Table 1).
Animal profiles
Bone X-ray and bone mineral density
Changes in body weight and HbA1c levels during the study are shown in Fig. 1. Body weight increased steadily in control mice throughout the 12-week observation period. In contrast, diabetic mice showed little gain in weight. Thus, the body weights of the diabetic animals were significantly (p < 0.01) reduced compared with the controls at the end of the experiment (Fig. 1A). HbA1c levels in the control group did not change throughout the experimental period. In contrast, HbA1c levels were significantly (p < 0.01) higher in the diabetic groups than in the control group during the experiment (Fig. 1B). These data indicate that hyperglycemia in diabetic mice was consistent and not transient. However, insulin treatment rescued these changes (Fig. 1).
No significant differences in bone shape could be detected between control, diabetic, and insulin-treated diabetic mice. However, diabetic mice showed severe osteopenia in the femora by X-ray (Fig. 2A). The BMD of the femora was decreased by about 15–20% in diabetic mice after 12 weeks of STZ treatment compared with that in the control mice (Fig. 2B). To assess the distribution of BMD in the femora, the bones were divided longitudinally into three equal parts, and the BMD of each part was measured (Fig. 2B). The BMD of each part was decreased to a similar extent in diabetic mice, suggesting that trabecular bone and cortex bone were equally affected by diabetic condition. Insulin treatment resolved the osteopenia and the reduction of BMD (Fig. 2).
Metabolic measurements
Bone histomorphometry
The metabolic measurements made at the end of the 12-week experimental period are summarized in Table 1. These data demonstrate that renal function was comparable in all three
Villanueva–Goldner staining indicated decreases of trabecular bone, stained green, and osteoid volume, stained red, in diabetic mice compared with control mice and insulin-treated
Fig. 5. TRAP stain and bone resorption parameters. The tibiae were removed after 12 weeks of STZ or vehicle treatment. The number of TRAP positive osteoclasts was lower in diabetic mice (B) than in control mice (A) and insulin-treated diabetic mice (C). Both bone resorption parameters were significantly decreased in diabetic group compared with other two groups (D, E). Control = control group; DM = diabetic group; Insulin = insulin-treated diabetic group. Magnification is 400×. Data are means ± S.D. *p < 0.05 vs. control group.
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diabetic mice (Figs. 3A–C). Histomorphometric measurements revealed that bone formation parameters [osteoid volume/ bone volume (OV/BV), osteoid surface/bone surface (OS/BS), and osteoid thickness (O.Th)] were significantly decreased in the diabetic group compared with the other two groups (Figs. 3D–F). Calcein double labeling showed that the width between the two stained labels was narrower in diabetic mice than in control mice and insulin-treated diabetic mice (Figs. 4A–C). The mineral apposition rate (MAR), reflecting the ability of individual osteoblasts to form bone, was about 50% decreased and consequently, the bone formation rate (BFR/BS), determined by the number and function of osteoblasts, was significantly lower in diabetic mice than in control mice and insulin-treated diabetic mice (Figs. 4D–F). Histological analysis of the proximal tibiae revealed that the number of TRAP-positive osteoclasts was lower in diabetic mice than in control mice and insulin-treated diabetic mice (Figs. 5A–C). Histomorphometric measurements supported the observation that bone resorption parameters [eroded surface (ES/BS) and osteoclast surface (Oc.S/BS)] were significantly lower in diabetic mice (Figs. 5D and E). The parameters for both bone formation and bone resorption were also significantly lower in diabetic mice, indicating that STZ-induced diabetic mice suppressed both bone formation and resorption, which is similar to that seen in humans.
Table 3 Correlations between the markers of oxidative stress and the histomorphometric parameters of bone formation OV/BV
OS/BS
O.Th
MAR
BFR/BS
r
r
r
r
r
Urinary 8-OHdG (ng/day) −0.721* − 0.864* − 0.447† − 0.815* − 0.893* Percentage of positive cells −0.725* − 0.877* − 0.464† − 0.818* − 0.94* (%) r is Spearman correlation coefficients. Data of control group, diabetic group, and insulin-treated diabetic group are combined for the analysis. *p value <0.01. †p value <0.05.
To assess the oxidative stress in bone tissues, immunohistochemical staining for 8-OHdG (Fig. 6) was performed. Staining of 8-OHdG was clearly intensified in the bone tissues, including osteoblasts, of diabetic mice (Figs. 6B, E) compared with control mice (Figs. 6A, D). In contrast, staining was prominently attenuated in insulin-treated diabetic mice (Figs. 6C, F). Moreover, the intensity of the staining in insulin-treated diabetic mice was similar to that in the control mice. Quantitative analysis confirmed these results (Table 2). Taken together, these results demonstrate that oxidative stress is increased not only in the whole body, but also in bone tissues, including the osteoblasts of diabetic animals. Moreover, the results demonstrate that insulin treatment rescues both lowturnover osteopenia and increased oxidative stress.
Assessment of oxidative stress Associations between oxidative stress and bone formation Urinary 8-OHdG, a sensitive indicator of oxidative DNA damage, was markedly (p < 0.01) higher in diabetic mice than in control mice. However, insulin-treated diabetic mice showed suppressed excretion of the marker, resulting in an absence of any significant difference with the control mice (Table 2).
Table 3 shows Spearman correlations of the markers of oxidative stress with the histomorphometric parameters of bone formation in all three groups combined. The markers of oxidative stress (urinary excretion of 8-OHdG and percentage
Fig. 6. Assessment of oxidative stress. Immunohistochemical staining for 8-OHdG revealed that staining to 8-OHdG was clearly intensified in bone tissues (B) including osteoblasts (E, arrowhead) of diabetic mice compared with control mice (A, D) and insulin-treated diabetic mice (C, F). A–C: Magnification is 200×. D–F: Magnification is 400×.
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of 8-OHdG) were inversely associated with the histomorphometric parameters of bone formation (OV/BV, OS/BS, O.Th, MAR, and BFR/BS). Discussion In the current study, our radiological analysis demonstrated a significant decrease in mouse trabecular bone volume (by about 15–20%) 12 weeks after the induction of diabetes, and our histological analysis confirmed that parameters for both bone formation (OV/BV, OS/BS, and BFR/BS) and resorption (ES/ BS and Oc.S/BS) were also significantly lower in STZ-induced diabetic mice. These results indicate that both bone formation and resorption are suppressed in STZ-induced diabetic mice, which simulate diabetic osteopenia. To exclude potential secondary effects of STZ treatment, so-called STZ toxicity, we also examined an insulin-treated diabetic group. Insulin treatment largely restored reduced BMD and prevented the suppression of both bone formation and resorption observed in the diabetic group, indicating that the osteopenia in STZinduced diabetic mice was strictly related to the diabetic state. In the present study, we selected the STZ-induced diabetic mouse from various animal models to characterize the phenotype of bone in diabetes. Although studies have examined the influence of diabetes on rat bone [20–22], few have directly addressed the influence of diabetes on the skeletal system of mice, an animal model that is acceptable for genetic manipulation. Consistent with rat studies [20–22] and human observations [1–3], we found a decrease in bone volume in the mouse model. Moreover, we demonstrated that the osteopenia in STZinduced diabetic mice exhibited low bone turnover. In the near future, these mice may be an adequate animal model to clarify the mechanisms responsible for low-turnover osteopenia as well as diabetic osteopenia through knockout and/or transgenic approaches. In vitro studies have reported that high glucose influences osteoblast differentiation [23], impairs bone formation [24], and inhibits bone mineralization [25]. In addition, experimental and clinical studies have demonstrated that osteoblast function is impaired under diabetic conditions [26,27]. Consistent with these reports, bone histomorphometry in the present study confirmed that osteoblast function and mineralization were impaired in STZ-induced diabetic osteopenia. Taken together, our results suggest that diabetic osteopenia without renal dysfunction is mainly caused by inhibition of osteoblast formation and function. Concerning the pathogenesis of osteoblast dysfunction, oxidative stress may be an important factor. In the present study, urinary excretion of 8-OHdG, a marker of oxidative DNA damage, was elevated in STZ-induced diabetic mice, while the marker was significantly suppressed to almost control levels in insulin-treated diabetic mice. Further immunohistological studies showed intensified immunostaining of the oxidative stress marker in bone tissue, including the osteoblasts, of diabetic mice, which was again diminished in insulin-treated diabetic mice. These findings indicate that oxidative stress in diabetic conditions was suppressed in insulin-treated diabetic
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mice accompanied by recovery from diabetic osteopenia. In vitro studies have shown that oxidative stress inhibits osteoblastic differentiation [15,16] and induces osteoblast insults and apoptosis [17,18]. However, there are few reports on the in vivo relationship between oxidative stress and diabetic osteopenia. The observations in the present study directly identify increased oxidative stress in diabetic bone as well as the whole body. In addition, we demonstrated that the markers of oxidative stress (urinary excretion of 8-OHdG and percentage of 8-OHdG positive cells) were inversely associated with the histomorphometric parameters of bone formation (OV/BV, OS/BS, O.Th, MAR, and BFR/BS). Although the present study did not fully clarify the main cause of diabetic osteopenia, our results suggest that the increase in oxidative stress may at least partly contribute to the development of diabetic osteopenia. To clarify the mechanism of the disease, further studies linking diabetic osteopenia to oxidative stress and bone turnover are planned. In conclusion, we demonstrated here that STZ-induced diabetic mice suppressed both bone formation and resorption associated with increased oxidative stress. These findings suggest that STZ-induced diabetic mice can be a useful animal model for diabetic osteopenia as seen in patients with diabetes. Furthermore, oxidative stress may be a therapeutic target in diabetic osteopenia. Acknowledgments This work is supported by the 21st Century COE Program “Center of Excellence for Signal Transduction Disease: Diabetes Mellitus as Model” awarded to M.K. by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We are grateful to the hard tissue research team at Kureha Chemical Co. for technical assistance. We also thank T. Nii-Kono and S. Matsuda (Kobe University Graduate School of Medicine) for their technical assistance. References [1] Kayath MJ, Dib SA, Vieira JG. Prevalence and magnitude of osteopenia associated with insulin-dependent diabetes mellitus. J Diabetes Complications 1994;8:97–104. [2] Kemink SA, Hermus AR, Swinkels LM, Lutterman JA, Smals AG. Osteopenia in insulin-dependent diabetes mellitus; prevalence and aspects of pathophysiology. J Endocrinol Invest 2000;23:295–303. [3] Lopez-Ibarra PJ, Pastor MM, Escobar-Jimenez F, Pardo MD, Gonzalez AG, Luna JD, et al. Bone mineral density at time of clinical diagnosis of adult-onset type 1 diabetes mellitus. Endocr Pract 2001 (Sep–Oct);7 (5):346–51. [4] Bouillon R. Diabetic bone disease. Calcif Tissue Int 1991;49:155–60. [5] Forsen L, Meyer HE, Midthjell K, Edna TH. Diabetes mellitus and the incidence of hip fracture: results from the Nord-Trondelag Health Survey. Diabetologia 1999;42:920–5. [6] Cozen L. Does diabetes delay fracture healing? Clin Orthop Relat Res 1972;82:134–40. [7] Herskind AM, Christensen K, Norgaard-Andersen K, Andersen JF. Diabetes mellitus and healing of closed fractures. Diabetes Metab 1992;18: 63–64. [8] Levin ME, Boisseau VC, Avioli LV. Effects of diabetes mellitus on bone mass in juvenile and adult-onset diabetes. N Engl J Med 1976;294:241–5. [9] Seino Y, Ishida H. Diabetic osteopenia: pathophysiology and clinical aspects. Diabetes Metab Rev 1995;11:21–35.
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