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Endocrinology Vol. 148, No. 1 198-205
Copyright © 2007 by The Endocrine Society

Bone Loss and Increased Bone Adiposity in Spontaneous and Pharmacologically Induced Diabetic Mice

Sergiu Botolin and Laura R. McCabe

Michigan State University, Departments of Physiology and Radiology, Molecular Imaging Research Center, East Lansing, Michigan 48824

Address all correspondence and requests for reprints to: Laura R. McCabe, Ph.D., Michigan State University, Departments of Physiology and Radiology, 2201 Biomedical Physical Science Building, East Lansing, Michigan 48824. E-mail: mccabel{at}msu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-dependent diabetes mellitus (IDDM) is associated with increased risk of osteopenia/osteoporosis in humans. The mechanisms accounting for diabetic bone loss remain unclear. Pharmacologic inducers of IDDM, such as streptozotocin, mimic key aspects of diabetes in rodents, allow analysis at the onset of diabetes, and induce diabetes in genetically modified mice. However, side effects of streptozotocin, unrelated to diabetes, can complicate data interpretation. The nonobese diabetic (NOD) mouse model develops diabetes spontaneously without external influences, negating side effects of inducing agents. Unfortunately, in this model the onset of diabetes is unpredictable, occurs in a minority of male mice, and can only be studied in a single mouse strain. To validate the relevance of the more flexible streptozotocin-induced diabetes model for studying diabetes-associated bone loss, we compared its phenotype to the spontaneously diabetic NOD model. Both models exhibited hyperglycemia and loss of body, fat pad, and muscle weight. Furthermore, these genetically different and distinct models of diabetes induction demonstrated similar bone phenotypes marked by significant trabecular bone loss and increased bone marrow adiposity. Correspondingly, both diabetic models exhibited decreased osteocalcin mRNA and increased adipocyte fatty acid-binding protein 2 mRNA levels in isolated tibias and calvaria. Taken together, multiple streptozotocin injection-induced diabetes is a valid model for understanding the acute and chronic pathophysiologic responses to diabetes and their mechanisms in bone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-DEPENDENT DIABETES mellitus (IDDM, type I diabetes) is a disease in which patients have little or no insulin secretion and hyperglycemia. The prevalence of IDDM is estimated to be 800,000 and growing in the United States. IDDM prevalence is also increasing in the adolescent/child population around the world (1, 2, 3, 4). Autoimmune, genetic, and environmental factors are some of the potential contributors involved in the development of IDDM. A confirmed fasting plasma glucose level greater than or equal to 126 mg/dl (previously 140 mg/dl) or confirmed nonfasting plasma glucose level greater than or equal to 200 mg/dl indicates a diagnosis of diabetes (5). Improved glucose monitoring and insulin delivery methods are allowing patients to live longer but also increasing their chance of complications as a result of extended chronic exposure to diabetic conditions. Complications that can dramatically affect life span and quality of life include: retinopathy, neuropathy, and nephropathy. More recently, IDDM has been associated with increased risk of osteopenia/osteoporosis (6, 7, 8, 9, 10, 11, 12, 13). Greater than 50% of male and female patients with IDDM exhibit bone loss compared with healthy age matched subjects (7). The mechanisms accounting for diabetic bone loss remain unclear.

A variety of animal models have been developed and used to examine the mechanisms of diabetes-associated complications, including spontaneous and pharmacologically induced diabetic rodent models. Streptozotocin and other pharmacologic inducers of diabetes (such as alloxan) play a key role in testing hypotheses related to mechanisms of diabetic complications. Streptozotocin enters insulin secreting pancreatic ß-cells through GLUT2 glucose transporters and causes DNA damage, depletion of nicotinamine adenine phosphate and ATP, and ultimately triggers pancreatic ß-cell necrosis (14). Previously, we and others have shown that streptozotocin-induced diabetes causes a significant decrease in bone formation and leads to bone loss in rats and mice (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Bone formation, rather than resorption, is defective in diabetic models as indicated by histomorphometry and serum markers of bone remodeling (6, 15, 21, 25, 26). Interestingly, we also found that streptozotocin-induced bone loss correlates with an increase in bone marrow adiposity (15), suggesting that cell maturation toward the adipocyte rather than osteoblast lineage could also contribute to the bone loss.

Multiple low-dose injections of streptozotocin can mimic key aspects of the development of diabetes (27, 28, 29). However, streptozotocin-induced diabetes does not require T or B cells (30) and can have cytotoxic effects on pancreatic ß-cells and influence other cell types in the body (31, 32, 33). For example, streptozotocin-induced diabetes can induce fat pad lipolysis in vivo, but can similarly induce lipolysis in isolated rat adipocytes in vitro (34), suggesting that in vivo effects could potentially be related to the streptozotocin treatment itself. Previously, we demonstrated that addition of streptozotocin to MC3T3-E1 osteoblast cultures did not affect gene expression or maturation (15), which is consistent with the inability of osteoblasts to take up streptozotocin because they do not express GLUT2. However, streptozotocin may influence other cells/tissues and impose streptozotocin-specific responses that could affect the phenotype of bone.

Nonobese diabetic (NOD) mice are susceptible to spontaneous development of autoimmune, type I insulin-dependent diabetes mellitus. The NOD mice have a unique MHC haplotype that contributes to the susceptibility to diabetes that can also be further modified by environmental conditions (housing, health, diet) (35). The NOD mice have been inbred for generations over the past 25 yr (36) and are one of the models of choice to examine diabetic complications because the mice do not require pharmacologic manipulation (35). Drawbacks of the NOD model include the unpredictable development of diabetes at different ages and only a minority of the mice develops diabetes (by 30 wk only 37% of NOD males become diabetic (37, 38), which makes large studies difficult and expensive. In addition, the NOD model is based on a single mouse strain unlike the streptozotocin diabetic model that can be used in a variety of genetically modified mice.

To determine whether or not the streptozotocin-induced diabetic model is a valid model for studying IDDM-associated bone loss, we compared the bone phenotype in diabetic NOD mice to the streptozotocin-induced diabetic model. Our findings demonstrate that similar to the streptozotocin-treated mice, diabetic NOD mice exhibit bone loss. In addition, we demonstrate that the bones of diabetic NOD mice have increased marrow adiposity, as determined histologically, and the mice exhibit altered gene expression profiles indicating a suppression of osteoblast markers and an induction of adipocyte markers. Taken together, our findings indicate that although reports suggest that streptozotocin itself can influence cells other than those of the pancreas, the streptozotocin-induced diabetic mouse model is a valid physiologic model to study the mechanisms contributing to diabetic bone loss.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Diabetic mouse models
Male NOD mice were purchased from Taconic Farms Inc. (Germantown, NY) at an age of 8 wk. Mice were housed in individual cages and maintained under pathogenic-free barrier conditions. Mice were screened for signs of diabetes by monitoring body weight and urine output. Mice that were confirmed as diabetic were maintained for another 4 wk before harvest. Age-matched euglcyemic mice were used as controls. Because of the spontaneity of diabetes, the age of mouse pairs (control and diabetic) ranged between 5 and 7 months. Therefore, in some analyses we examined changes between diabetic mice and their age matched, paired control mice, to avoid the added variation of changes associated with aging.

Pharmacologic induction of type I diabetes in adult male BALB/c mice (Harlem Laboratories, Houston, TX) was performed by ip injection with streptozotocin (40 µg/g body weight in 0.1 M citrate buffer), a pancreatic ß-cell cytotoxin, for 5 d (14, 39). Controls were injected with buffer alone. Seven days after the last injection, nonfasting blood glucose measurements were performed using blood obtained from the lateral saphenous vein and a glucometer (Accu-Check instant, Roche Molecular Biochemicals Corp., Indianapolis, IN). Mice with blood glucose levels greater than 300 mg/dl were considered diabetic. Animals were euthanized 4 wk after confirmation of diabetes.

All mice were kept on a 12-h light, 12-h dark cycle at 23 C and received food (standard lab chow) and water ad libitum. After euthanasia, tibiae were immediately removed, freed from soft tissue, and either fixed in formalin [for histology and microcomputed tomography (µCT) analyses] or snap-frozen in liquid nitrogen and stored at –80 C (for RNA analyses). Tibialis anterior muscles and sc femoral fat pads were dissected from surrounding tissues and weighed. Animal studies were conducted in accordance with the Michigan State University All-University Committee on Animal Use and Care.

Plasma measurements
Blood was obtained from mice at the time of euthanasia and blood serum prepared from each sample by centrifugation for 5 min at 3000 rpm. Serum was stored frozen at –20 C. Glucose concentration in serum samples was determined using a glucose assay kit (Sigma, St. Louis, MO). Serum osteocalcin levels were measured using Mouse Osteocalcin EIA Kit (Biomedical Technologies Inc., Stoughton, MA) according to the manufacturer’s instructions. Serum PYD was measured using Metra PYD kit (Quidel Corp., San Diego, CA) according to the manufacturer’s instructions. Quantitative determinations of serum glycerol, total triglyceride and true triglyceride (expressed as equivalent triolein concentration) levels were performed using a Serum Triglyceride Determination Kit (Sigma).

RNA analysis
Whole tibiae and calvaria halves were crushed under liquid nitrogen conditions using a Bessman Tissue Pulverizer and RNA extracted using the method of Chomczynski and Sacchi as previously described (40, 41). RNA integrity was verified by formaldehyde-agarose gel electrophoresis. Synthesis of cDNA was performed by RT with 4 µg of total RNA using the Superscript II kit with oligo(deoxythymidine)(12–18) primers as described by the manufacturer (Invitrogen, Carlsbad, CA). cDNA (1 µl) was amplified by PCR in a final volume of 25 µl using the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) with 10 pmol of each primer (Integrated DNA Technologies, Coralville, IA). Osteocalcin was amplified using 5'-ACG GTA TCA CTA TTT AGG ACC TGT G-3' and 5'-ACT TTA TTT TGG AGC TGC TGT GAC-3' (42). Runx2 was amplified using 5'-GAC AGA AGC TTG ATG ACT CTA AAC C-3' and 5'-TCT GTA ATC TGA CTC TGT CCT TGT G-3' (43). Peroxisome proliferator-activated receptor (PPAR) {gamma}2 was amplified using 5'-TGA AAC TCT GGG AGA TTC TCC TG-3' and 5'-CCA TGG TAA TTT CTT GTG AAG TGC-3' (44). Adipocyte fatty acid-binding protein 2 (aP2) was amplified using 5'-GCG TGG AAT TCG ATG AAA TCA-3' and 5'-CCC GCC ATC TAG GGT TAT GA-3' (45). Cathepsin K was amplified using 5'-GCA GAG GTG TGT ACT ATG A-3' and 5'-GCA GGC GTT GTT CTT ATT-3' (46). Alkaline phosphatase was amplified using 5'-CGT AAT CTA CCA TGG AGA CAT TTTC-3' and 5'-GAC TGT GGT TAC TGC TGA TCA TTC-3' (15). Cyclophilin, which was not modulated under diabetic conditions, was used as a control for RNA levels; it was amplified using 5'-ATT CAT GTG CCA GGG TGG TGA C-3' and 5'-CCG TTT GTG TTT GGT CCA GCA-3' (47, 48). Real-time PCR was carried out for 40 cycles using the iCycler (Bio-Rad), and data were evaluated using the iCycler software. Each cycle consisted of 95 C for 15 sec, 60 C for 30 sec (except for runx2 and osteocalcin, which had an annealing temperature of 65 C) and 72 C for 30 sec. RNA-free samples, a negative control, did not produce amplicons. Melting curve and gel analyses (sizing, isolation, and sequencing) were used to verify single products of the appropriate base pair size.

Tissue and bone histology and histomorphometry
Proximal tibiae isolated from control and diabetic mice were fixed in 10% neutral buffered formalin. Fixed samples were processed on an automated Thermo Electron Excelsior tissue processor for dehydration, clearing and infiltration using a routine overnight processing schedule. Samples were then embedded in Surgipath embedding paraffin on a Sakura Tissue Tek II embedding center. Paraffin blocks were sectioned at 5 µm on a Reichert Jung 2030 rotary microtome. Slides were stained with hematoxylin and eosin. Visible adipocytes, greater than 30 µm, were counted in the trabecular region ranging from the growth plate to 2 mm away distally.

µCT analysis
Fixed tibias were scanned using a GE Explore Locus µCT system at a voxel resolution of 20 µm obtained from 720 views. Beam angle of increment was 0.5 and beam strength was set at 80 peak kV and 450 µA. Each run included control and diabetic bones and a calibration phantom to standardize grayscale values and maintain consistency. Based on autothreshold and isosurface analyses of multiple bone samples, a fixed threshold (1400) was used to separate bone from bone marrow. Cortical bone analyses were made in a defined 3-mm3 cube in the mid-diaphysis 1 mm proximal of the tibial-fibular junction. Trabecular bone analyses were done in a region of trabecular bone defined at 0.17 mm (~1% of the total length) under the growth plate of the proximal tibia extending 2 mm toward the diaphysis, and excluding the outer cortical shell. Bone mineral content, mineral density, and volume fraction values were computed by a GE Healthcare MicroView software application for visualization and analysis of volumetric image data.

Statistical analysis
All statistical analyses were performed using Microsoft Excel data analysis program for Student’s t test analyses. Values are expressed as means ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Male NOD mice (starting at the age of 2 months) were screened for signs of diabetes by monitoring body weight and urine output. Detection of diabetes occurred in mice between 5 and 7 months of age, as predicted based on previous reports (35). After confirmation of diabetes, mice were maintained for an additional 4 wk to address the effect of chronic diabetic conditions on bone phenotype. Age-matched euglycemic littermate mice were used as controls. Diabetic NOD mice exhibit elevated serum glucose levels similar to the pharmacologically induced streptozotocin-diabetic mouse model (Fig. 1Go). Diabetic NOD mice lost more weight than streptozotocin-induced diabetic mice; however, control NOD mice were significantly heavier than the control BALB/c mice in the streptozotocin experiment (Fig. 1Go). Consistent with a significant loss of body weight in the NOD mice, fat pad and muscle weights were also significantly reduced and by a greater extent when compared with streptozotocin-induced diabetic mice (Fig. 1Go). Again, consistent with greater body mass, control NOD mice had greater fat pad and muscle mass than control mice in the streptozotocin experiment.


Figure 1
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  		FIG. 1. Spontaneously diabetic NOD mice and streptozotocin-induced diabetic mice exhibit similar decreases in body, fat pad and muscle weights. Body parameters were examined in control (white bars) and diabetic (for 4 wk, black bars) adult NOD mice and streptozotocin-induced diabetic mice (STZ) between 6 and 8 months of age. Values are means ± SE. *, P < 0.05.

 
To address the effect of spontaneous type I diabetes on bone mass, µCT was used to image and quantitate bone densities in control and diabetic mice. Representative images of proximal tibias from control and diabetic NOD and streptozotocin models demonstrate reduced trabecular bone density under the growth plate region of the bones (Fig. 2Go). Because diabetes in NOD mice occurs at random ages and age may influence bone parameters, bone measurements were expressed as a percentage change relative to age-matched littermate controls. Quantitation of bone mineral content, bone mineral density, and bone volume fraction (BVF) indicate that diabetes induces a significant reduction in all parameters by a similar extent in NOD and streptozotocin diabetic mice (Table 1Go). Reductions were also seen in cortical bone, but did not reach statistical significance (Table 1Go), except in the streptozotocin BVF parameter.


Figure 2
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  		FIG. 2. Spontaneously diabetic NOD mice and streptozotocin-treated mice exhibit a similar degree of bone loss. Shown are representative µCT images of proximal tibias isolated from control and diabetic (for 4 wk) adult NOD mice and streptozotocin-induced diabetic mice (STZ) between 6 and 8 months of age.

 

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  		TABLE 1. Bone parameters (percentage decrease; diabetic vs. control)

 
Previously, we reported that streptozotocin-induced diabetes is associated with increased bone marrow adiposity; therefore, we examined whether a spontaneously diabetic mouse model would also exhibit this phenotype or whether streptozotocin itself was somehow inducing marrow adiposity. Representative histological images of bone marrow sections (region located beneath the growth plate) demonstrate an increased number of visible adipocytes in NOD and streptozotocin diabetic mice compared with their control counterparts (Fig. 3AGo). Actual counting of adipocytes within this region demonstrated that control NOD mice tended to have a greater number of adipocytes when compared with citrate buffer injected control animals from the streptozotocin study. However, in both cases, induction of diabetes increased marrow adipocyte number (Fig. 3BGo). On average, the number of adipocytes in the diabetic NOD mice was greater than the number in streptozotocin-induced diabetic mice, but the fold increase above control levels was greater in the streptozotocin diabetic mice (3- to 4-fold vs. 8-fold). This is in part due to the low number of adipocytes in the control mice in the streptozotocin study.


Figure 3
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  		FIG. 3. Increased bone marrow adiposity in spontaneously diabetic NOD mice is similar to changes seen in streptozotocin-induced diabetic compared with control mice. A, Representative histological images are shown of proximal tibias isolated from control and diabetic (4 wk) NOD mice and streptozotocin-induced diabetic mice (STZ) between 6 and 8 months of age. Sections are stained with hematoxylin and eosin. B, Quantitation of adipocytes in control (white bars) vs. diabetic (black bars) NOD and streptozotocin (STZ)-injected mouse tibias from the growth plate to 2 mm beneath the growth plate. Values are averages ± SE. *, P < 0.05.

 
Confirmation of increased adiposity was obtained through gene analysis studies. PPAR{gamma}2 and aP2 are known markers of adipocyte differentiation and maturation, so we examined their levels in RNA isolated from control vs. diabetic tibias. Both PPAR{gamma}2 and aP2 mRNA levels are elevated in diabetic NOD mice (Fig. 4Go). The fold induction was even greater in diabetic NOD mice than what was seen with the streptozotocin model (3- to 5-fold vs. 0.5- to 2-fold, respectively). Elevated adipocyte gene expression in the NOD mice is consistent with the greater adipocyte number in the NOD (control and diabetic) compared with streptozotocin (control and diabetic) mice (Fig. 3BGo).


Figure 4
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  		FIG. 4. Adipocyte markers are increased, whereas mature osteoblast markers are decreased in diabetic NOD and streptozotocin-injected (STZ) mice compared with controls. Total RNA was extracted from tibia isolated from control (C, white bars) and diabetic (D, black bars) NOD mice and control and streptozotocin-induced diabetic mice 4 wk after the confirmation of diabetes. Levels of aP2, PPAR{gamma}2, osteocalcin (OC), and Runx-2 mRNAs were determined by RT-PCR and are expressed relative to cyclophilin, a nonmodulated, housekeeping gene. For NOD studies, gene expression in diabetic mice was compared with levels in age-matched control mice, which were set to 1. Values are averages ± SE. *, P < 0.05.

 
In contrast to genes associated with adiposity, expression of a marker of osteoblast maturation, osteocalcin, was significantly decreased in diabetic NOD mice (Fig. 4Go). Again, the suppression was a greater fold decrease than what was observed in the streptozotocin model (~5- vs. 2-fold, respectively). Suppression of osteocalcin mRNA levels, and hence osteoblast maturation and/or number, is consistent with decreased bone density in diabetic NOD and streptozotocin mice. Interestingly, in these studies, runx2, an early marker of osteoblast lineage selection and osteoblast differentiation was not suppressed in diabetic NOD or streptozotocin-induced mice. Analyses of another marker of osteoblast maturation, alkaline phosphatase, also did not show a change between control and diabetic animals in either model of diabetes (Fig. 5Go).


Figure 5
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  		FIG. 5. Cathepsin K and alkaline phosphatase (Alk Phos) mRNA levels are not modified by diabetic conditions in either NOD or streptozotocin-induced mouse models. Total RNA was extracted from tibia isolated from control (C) and diabetic (D) NOD mice and streptozotocin-induced diabetic mice 4 wk after the confirmation of diabetes. Levels of cathepsin K and alkaline phosphatase (Alk Phos) mRNAs were determined by RT-PCR and are expressed relative to cyclophilin, a nonmodulated, housekeeping gene. For NOD studies, gene expression in diabetic mice was compared with levels in age-matched control mice, which were set to 1. Values are averages ± SE.

 
Although osteoclast activity and number have been reported to be unaltered in diabetes, we wanted to test whether a similar phenotype occurred in the spontaneously diabetic NOD model. Levels of cathepsin K mRNA in control and diabetic NOD mouse tibias were similar (Fig. 5Go) and corresponded with findings in streptozotocin-induced diabetic mice (Fig. 5Go).

To determine whether the induction of aP2 and suppression of osteocalcin mRNA levels are specific to endochondrial bones such as the tibia, we examined their expression in an intramembranous bone, the calvaria. Figure 6Go demonstrates that aP2 mRNA levels are increased in calvaria from diabetic mice of either model system, spontaneous or pharmacologically induced. Consistent with a reciprocal relationship, osteocalcin mRNA levels were decreased in calvaria from diabetic mice of either model system (Fig. 6Go). These results directly correspond with results from tibial RNA (Fig. 4Go).


Figure 6
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  		FIG. 6. Changes in mRNA levels seen in tibia are also seen in calvaria. Total RNA was extracted from calvaria isolated from control (C, white bars) and diabetic (D, black bars) NOD mice and control and streptozotocin-induced diabetic mice 4 wk after the confirmation of diabetes. Levels of aP2 and osteocalcin (OC) mRNAs were determined by RT-PCR and are expressed relative to cyclophilin, a nonmodulated, housekeeping gene. For NOD studies, gene expression in diabetic mice was compared with levels in age-matched control mice, which were set to 1. Values are averages ± SE. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many models of type I diabetes exist that involve pharmacologic induction or that genetically favor spontaneous induction of diabetes (49). Diabetes induced by multiple low-dose streptozotocin injection allows accurate monitoring of physiologic changes at the onset of diabetes (before hyperglycemia) and is thought to cause pancreatic ß-cell destruction that in many ways is similar to that seen with the onset of type I diabetes in humans (27). On the other hand, the NOD mouse model provides spontaneously diabetic mice without the use of pharmacologic agents, thereby negating any potential side effects of the diabetes-inducing agent. Of the few studies that compared phenotypes between NOD and streptozotocin models, sympathetic neuropathy (50), hypercapnia ventilatory responses (51), and healing and neovascularization (52) were suppressed in both models, validating the utility of the streptozotocin model for studying the mechanisms accounting for these complications. Here we examined the validity of the streptozotocin model for studying diabetes-associated bone complications. We compared the bone phenotype of NOD vs. streptozotocin diabetic mice and demonstrate that the general and bone-specific responses to type I diabetes were nearly identical.

Comparison of gene expression in isolated bones from control and diabetic mice of NOD and streptozotocin models demonstrated in both models, a switch from genes associated with a mature osteoblast phenotype to genes associated with an adipocyte phenotype. Specifically, expression of a marker of mature osteoblasts, osteocalcin, was suppressed, whereas adipocyte markers, PPAR{gamma}2 and aP2, were induced by diabetic conditions. Responses in diabetic NOD mice were stronger than BALB/c mice and could stem from genetic differences as discussed later. Our findings of bone loss and decreased markers of osteoblast maturation in the basal state of diabetic NOD mice corresponds with a recent report by Thrailkill et al. (53) demonstrating reduced bone healing in diabetic compared with euglycemic NOD mice in response to distraction osteogenesis. Interestingly, runx2 and alkaline phsosphatase mRNA levels, markers of osteoblast lineage selection and early and mid-stage osteoblast maturation, were not affected in NOD or streptozotocin-induced diabetic mice, suggesting that later stages of osteoblast maturation may be specifically influenced by type I diabetes. Previously, we observed a similar finding in mice confirmed diabetic for 4 wk (15). More recently, we determined that runx2 mRNA levels are suppressed at early time points [d 7 and 14 after confirmation of diabetes (54)]. Similarly, in a bone marrow ablation model, streptozotocin-induced diabetes suppressed runx 2 mRNA levels at 4 and 6 d after ablation, whereas at 16 d after ablation levels were restored to normal (22). The variability of runx2 expression in diabetic models may stem from its involvement in a variety of bone-related functions including early cartilage lineage pathway, osteoblast lineage selection, and osteoblast maturation, as well as the ability of a variety of factors/stresses to readily modulate runx2 mRNA levels (42, 55, 56, 57, 58).

Our findings also indicate that intramembranous bone exhibits a similar phenotypic change in response to diabetic conditions. Specifically, aP2 mRNA levels were up-regulated and osteocalcin mRNA levels were down-regulated in diabetic calvaria. These findings directly parallel the changes that we observed in the tibia. Although the total marrow area of the calvaria is smaller than that of the tibia, the amount of total bone area is also smaller in the calvaria compared with the tibia. Thus, our finding that changes in aP2 and osteocalcin expression occur in the calvaria is not surprising and does not rule out a role for changes in occurring in marrow precursor cells. In addition, the changes that we observed in calvarial gene expression are consistent with calvarial histology and density changes in streptozotocin-induced diabetic compared with control mice (Martin, L. M. and L. R. McCabe, manuscript in preparation). Our findings are also consistent with other studies demonstrating that diabetic conditions influence the calvaria. Specifically, it has been reported that the receptor for advanced glycation end products is expressed at higher levels in diabetic compared with control calvaria and that craniotomy defects in diabetic animals require a greater healing time compared with controls (59).

Previous studies have indicated that multiple low-dose streptozotocin-induced diabetes can induce fat pad lipolysis and hyperlipidemia (60), similar to our findings of reduced body weight and fat pad weights in our streptozotocin experiment. However, studies also suggest that streptozotocin alone can stimulate lipolysis in vitro (34). Our studies indicate that fat pad lipolysis also occurs in NOD mice, in the absence of streptozotocin indicating that the loss peripheral fat pad mass is associated with type I diabetic conditions and not a secondary effect of streptozotocin treatment. In contrast to the significant lipolysis of peripheral fat stores, we found that the bone marrow exhibited signs of lipogenesis in diabetic NOD mice similar to the streptozotocin-induced diabetic mice (15). These findings demonstrate that bone adiposity is not linked to peripheral fat pad adiposity. Differences in local cytokine and growth factor levels (perhaps due to differences in local cell types such as hematopoeitic cells) may contribute to the dissociated regulation. Although few studies have examined the link between marrow and peripheral adiposity, several studies have determined an association between bone loss and increased bone marrow adiposity, which is evident in age-related bone loss (61, 62, 63) and unloading of bone (limb disuse) (64) and is seen in vitro when adipocytic signals reduce the number of mature bone producing osteoblast cells (65, 66, 67, 68).

As with any study, the role of mouse strain could confound responses through genetic differences. Specifically, mouse strains have been demonstrated to influence basal bone parameters such as bone mineral density and bone volume fraction (69). Actual basal values of bone parameters in euglycemic, control mice (not shown), were lower in the NOD compared with the BALB/c mice. In particular, tibial trabecular bone volume fraction was nearly 50% lower in control euglycemic NOD mice compared with BALB/c mice (21 ± 3% vs. 41 ± 2%, respectively). Other studies have demonstrated that basal bone parameters, particularly those of the tibial trabecular region, can vary more than 4-fold between strains (70). In addition, it is also known that the magnitude of a bone response to a stimuli or stress can be influenced by mouse strain as demonstrated in mouse models of disuse (71), loading (72) or ovariectomy (70). However, this was not the case in our study. Specifically, diabetes induced significant losses (by 26–50%) in all trabecular bone parameters (bone mineral content, bone mineral density, BVF) that were nearly of identical magnitude in NOD and BALB/c mice. Both strains also exhibited a trend toward suppression (by 3–16%) of all cortical bone parameters. Only the BALB/c strain exhibited statistically significant suppression of cortical BVF, but it is likely that with extended diabetes the suppression of the NOD cortical bone volume fraction would also reach statistical significance. Our past studies consistently demonstrate that trabecular bone is more susceptible to diabetic bone loss than cortical bone (15, 54); this is likely due to the trabecular region having a higher remodeling rate and the intimate association of the trabecular bone with the marrow environment, cytokines, and growth factor stimuli (73).

Our findings are the first to demonstrate that effects on bone phenotype are similar between two genetically different and distinct models of diabetes induction: spontaneously diabetic NOD mice vs. streptozotocin-induced diabetic mice. Therefore, our studies confirm that bone loss and increased marrow adiposity are hallmarks of the diabetic bone phenotype. Although diabetes occurs in NOD mice without external influences, the streptozotocin model allows accurate timing of the onset of diabetes, readily induced diabetes in mice, and exhibits many of the parameters associated with the onset of IDDM in humans. Taken together, multiple streptozotocin injection-induced diabetes is a valid model for understanding the acute and chronic pathophysiologic responses to diabetes and their mechanisms in bone.


   Acknowledgments
 
We thank Regina Irwin for her technical assistance and contributions to the RNA analyses and the Investigative Histology Laboratory in the Department of Physiology, Division of Human Pathology at Michigan State University for their expertise and assistance with the histology analyses. We also thank members of the McCabe lab: Lindsay Martin, Katherine Motyl, and Andrea Thelen for their contributions to and critical reading of this manuscript.


   Footnotes
 
This work was funded by National Institutes of Health Grant DK061184 (to LRM.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 19, 2006

Abbreviations: aP2, Adipocyte fatty acid-binding protein 2; BVF, bone volume fraction; µCT, microcomputed tomography; IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; PPAR, peroxisome proliferator-activated receptor.

Received July 27, 2006.

Accepted for publication October 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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