Elsevier

Bone

Volume 44, Issue 4, April 2009, Pages 648-655
Bone

Type 2 diabetic mice demonstrate slender long bones with increased fragility secondary to increased osteoclastogenesis

https://doi.org/10.1016/j.bone.2008.12.012Get rights and content

Abstract

Type 2 diabetics often demonstrate normal or increased bone mineral density, yet are at increased risk for bone fracture. Furthermore, the anti-diabetic oral thiazolidinediones (PPARγ agonists) have recently been shown to increase bone fractures. To investigate the etiology of possible structural and/or material quality defects, we have utilized a well-described mouse model of Type 2 diabetes (MKR). MKR mice exhibit muscle hypoplasia from birth with reduced mass by the pre-diabetic age of 3 weeks. A compensatory hyperplasia ensues during early (5 weeks) development; by 6–8 weeks muscle is normal in structure and function. Adult whole-bone mechanical properties were determined by 4-point bending to test susceptibility to fracture. Micro-computed tomography and cortical bone histomorphometry were utilized to assess static and dynamic indices of structure, bone formation and resorption. Osteoclastogenesis assays were performed from bone marrow-derived non-adherent cells. The 8-week and 16-week, but not 3-week, male MKR had slender (i.e., narrow relative to length) femurs that were 20% weaker (p < 0.05) relative to WT control femurs. Tissue-level mineral density was not affected. Impaired periosteal expansion during early diabetes resulted from 250% more, and 40% less of the cortical bone surface undergoing resorption and formation, respectively (p < 0.05). Greater resorption persisted in adult MKR on both periosteal and endosteal surfaces. Differences were not limited to cortical bone as the distal femur metaphysis of 16 week MKR contained less trabecular bone and trabecular separation was greater than in WT by 60% (p < 0.05). At all ages, MKR marrow-derived cultures demonstrated the ability for enhanced osteoclast differentiation in response to M-CSF and RANK-L. Taken together, the MKR mouse model suggests that skeletal fragility in Type 2 diabetes may arise from reduced transverse bone accrual and increased osteoclastogenesis during growth that is accelerated by the diabetic/hyperinsulinemic milieu. Further, these results emphasize the importance of evaluating diabetic bone based on morphology in addition to bone mass.

Introduction

Diabetes is an emerging epidemic that has consequences for musculo-skeletal growth and development, and bone fracture risk [1], [2], [3], [4], [5], [6], [7], [8]. Yet, Type 2 diabetics (non-insulin-dependent, T2D) often demonstrate increased bone mineral density (BMD) [9], [10], [11], [12], [13]. Furthermore, the use of anti-diabetic oral thiazolidinediones (PPARγ agonists) in T2D has recently been shown to increase fracture risk in the absence of a reduction in BMD [14]. These apparent paradoxes suggest that the increased bone fragility in T2D may not be discerned from bone mass, but may depend on aspects of bone structure or tissue quality that are not revealed from BMD. A number of factors have been postulated to be etiologically associated with poor bone accrual in diabetes, including an insulin-related direct reduction in bone formation and reduced force generating capacity of muscle.

Studies of bone turnover in animal models are most accurately assessed by histomorphometry. Several models have demonstrated diabetes-induced alterations in bone and could aid in elucidating the cellular and biomechanical mechanisms that may lead to increased risk of fragility fractures [15], [16], [17], [18], [19], [20]. Models for Type 1 diabetes (T1D) are most prevalent and have demonstrated decreased breaking strength of femoral shafts when compared to controls [21], [22]. However, many of these are drug-induced models confounded by side-effects that can result in significant health issues. For example, the pancreatic β-cell toxins streptozotocin or alloxan have been injected into both rats and mice to induce an uncontrolled diabetic state [23], [24]. Growth trajectories are immediately altered such that diabetic animals fail to grow (i.e., the rates of long bone growth and body mass gains are significantly stunted) [25], [26]. However, stunted longitudinal growth is not normally associated with diabetes. Most recently, using a model of streptozotocin-induced insulinopenia, Hamada et al. confirmed this affect on growth as well as a reduction in trabecular bone volume with reduction in both bone formation and bone resorption in young animals, changes that were reversed by insulin replacement therapy [25]. The main conclusion of that study was that in T1D, insulinopenia is the primary etiology for osteopenia, though secondary mechanisms, such as oxidative stress, may also play a role [25]. The cellular mechanisms behind these differences require further examination.

Models of T2D have thus far been less biologically insightful because many induce obesity (another state of insulin resistance) or are limited to rats [27], [28], [29], [30]. There is limited evidence for increased fracture risk due to greater levels of obesity and obesity may even protect a subject from fracture, despite being associated with lower physical activity [3]. Obesity increases the load-bearing forces during locomotion and, absent a bone formation defect, elicits structural compensation, i.e., more weight requires a larger frame [31]. Obesity may also enhance endogenous production of estrogen, a hormone important for bone cell regulation in both sexes [32]. Non-obese T2D occurs spontaneously in rats such as the Torii, which was outbred from Sprague–Dawley in 1997, and also exhibits stunted longitudinal bone growth; however, the disease has unknown etiology and is associated with insulinopenia [28].

We have generated a non-obese transgenic mouse model of T2D (MKR mice) by blocking both the insulin and IGF-signaling pathways specifically in skeletal muscle. Blocking of both receptors in muscle abrogates downstream signaling, particularly the AKT pathway, which regulates glucose uptake into muscle. MKR mice are born with naturally occurring hyperinsulinemia and insulin resistance in muscle. Early in life (∼ 2 weeks) the MKR mice develop dyslipidemia, and the mice eventually develop frank diabetes with hyperglycemia (∼ 7–8 weeks) [33], [34]. This scenario of disease development is similar to the human disease, where insulin resistance and hyperinsulinemia, with dyslipidemia eventually progress to diabetes. The MKR mouse model is useful since no exogenous agents are required to induce disease, thus removing potential confounding aspects. In addition, MKR mice are non-obese, excluding another confounding variable. In this study we employed the MKR mouse model to examine the effect of progression of T2D on skeletal integrity. Skeletal characterization was performed at the pre-diabetic (3 weeks of age), early diabetic (8 weeks) and established diabetic stages (16 weeks). Here we demonstrate that early onset of insulin resistance does not affect longitudinal growth but alters transverse expansion of skeletal structures, which worsens with the progression to the adult diabetic state.

Section snippets

Materials and methods

The generation of the MKR Type 2 diabetic mouse has been described elsewhere [33]. MKR male mice, on a FVB/N background, were bred to homozygosity, and were compared to wild-type FVB/N controls (WT). MKR mice demonstrate severe insulin resistance starting at birth, hyperinsulinemia and hyperlipidemia at 3–4 weeks of age but with normoglycemia [33]. At 6–8 weeks of age MKR mice develop diabetes with blood glucose levels of 250–400 mg/dl versus 130–160 mg/dl for WT. The mice were kept on a 12-h

Diabetes phenotype of MKR mice

Male MKR and FVB/N control mice (WT) were weaned at 3 weeks of age and following genotyping as previously described were monitored weekly for blood glucose levels [33]. All mice remained normoglycemic from 3 weeks to 5 weeks of age. By 7 weeks of age, fed blood glucose levels in MKR mice rose to diabetic levels (between 250 and 400 mg/dl), whereas WT remained normoglycemia (between 130 and 160 mg/dl). The hyperglycemia of the MKR mice remained at 16 weeks of age. Hyperlipidemia and

Discussion

Fragility fractures are associated with both Types 1 and 2 diabetes despite the fact that bone mass (BMD) losses do not occur in Type 2 diabetes (T2D) [9], [10], [11], [12], [13]. The reductions in BMD with Type 1 diabetes (T1D) occur early in the disease process and are associated with increased bone fragility and fractures later in life. While the reductions in BMD in T1D have been well established, the cellular cause(s) have not been defined completely, with both increased bone turnover and

Conflict of interest statement

The authors have nothing to disclose.

Acknowledgments

We are indebted to Ken Inagaki, Damien Laudier, Valerie Williams, David Berman, and Bin Hu for their help on this project. Financial support received from funding agencies in the United States including the Mount Sinai School of Medicine (Steckler Fund Grant to DL), NIAMS (NIH Grants AR41210 to MBS, AR44927 to KJJ and AR054919 and AR055141 to SY), National Space Biomedical Research Institute (NASA Grant NCC 9-58 to MBS) and the Charles H. Revson Foundation (Fellowship to JCF). The statements

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