Fragility fractures are associated with both Type 1 and 2 diabetes despite the fact that bone mass (BMD) losses do not
occur in Type 2 diabetes (T2D) [9
]. 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 decreased osteoblastic activity implicated in the net bone loss [43
]. In contrast, there is no conspicuous reduction in BMD in T2D [10
]. Thus, the increase in fragility fractures in T2D is more likely the result of changes in bone integrity and/or quality. Indeed, our biomechanical studies in the hyperglycemic MKR mice support this concept, with significantly altered relative fracture toughness (as measured from post-yield deflection) and reduced overall bone stiffness and strength in the diseased state. Mechanical data from other T2D models are unavailable for comparison. The observed changes in stiffness and strength in these studies are largely accounted for by the changes in morphology and consistent with the smaller cross-sectional sizes (Tt.Ar and J) of MKR bone compared to WT. That MKR femurs are more slender (narrow relative to length) is an important finding because slenderness in human bone has been shown to be a significant indicator of fracture risk [44
We have now identified both muscle and bone deficiencies in the MKR mouse albeit first presenting at different ages. The MKR mouse exhibits muscle hypoplasia with a 20% lower protein content in soleus and extensor digitorum longus early in life at 3 weeks of age. Interestingly, by 5–6 weeks, compensatory mechanisms are at work as shown recently by skeletal muscle hypertrophy and strengthening [34
]. Therefore, even before frank diabetes appears at ~7–8 weeks, skeletal muscle approximates that of WT but with protein content that is 15% higher in MKR than in WT mice at 5 weeks of age [34
]. Thus, it is conceivable that the bone structural effects seen at 8 and 16 weeks of age arose early in growth when the effects of hypoplastic skeletal muscle may affect bone structure. However, despite the muscle deficit there was no significant difference in femoral cortical bone characteristics between MKR and WT mice at 3 weeks of age. Moreover, as insulin resistance in the MKR mice progressed and skeletal muscle function improved at 8 and 16 weeks, MKR mice demonstrated significant structural impairments in skeletal integrity compared to WT mice, reflected by reduced cortical bone size and trabecular bone volume. This strongly argues that the progression of the MKR mice to the Type 2 diabetic state played a role in these bone structural changes.
Hyperglycemia and hyperinsulinemia have been postulated as possible causes of skeletal changes in diabetic patients. While the mechanism(s) are not known, advanced glycosylation end products (AGEs) working through their specific receptors (RAGE) [19
] have been postulated to alter bone cell function. Hyperglycemia suppresses osteoblast proliferation and differentiation in culture systems, and the mechanisms may involve expression of c-jun, a transcription factor that affects collagen expression in bone-derived cell lines [50
]. Insulin is aparently a hormone important for osteoblast function, chondrogenesis and collagen synthesis, and may also be involved in osteoclastogenesis [52
]. Fracture risk has been shown to increase with insulin use [7
]. However, in mouse models of insulinopenia (T1D), reduced serum osteocalcin and lower bone osteoblast numbers are associated with reduced osteoid and reduced bone mineral accretion rates, resulting in lower bone turnover rates. In a model of T2D, a knockout mouse for the insulin receptor (IR), BMD was normal, possibly compensated for by the IGF-1R or perhaps demonstrating a lesser role of the insulin resistance in bone formation [55
Other features of the T2D milieu beyond AGEs are yet to be fully studied regarding their impact on bone, such as lipids and oxidative stress. In two separate models, one a streptozotocin diabetic mouse and one a rat model of non-obese, insulin-sensitive T2D, osteopenia was demonstrated. In both models oxidative stress markers were measured and found to be elevated and normalized with insulin therapy. In the former model, the oxidative stress marker (8-OHdG) was also seen to be increased in bone by immunohistochemical analysis, and the authors speculated that the inhibition of osteoblastic differentiation could be explained by this effect [25
]. Indeed, oxidative stress may affect osteoblastic differentiation as demonstrated in vitro [56
]. In our laboratory, MKR mice also demonstrate increased oxidative stress markers in serum and liver (unpublished), and this potential pathogenic factor and its effect on bone formation and bone turnover requires more extensive study.
The principal bone cell defect in MKR mice is increased osteoclastogenesis, particularly at later ages, as demonstrated in the current study both by histomorphometry as well as bone marrow cultures. The slender bones in the MKR diaphyses, as well as the reduced cancellous bone volume and increased trabecular separation appear to result principally from increased osteoclastic activity and a small transient suppression of osteoblastic activity. That marked increases in cell differentiation were present in vitro, in the absence of the circulating endocrine milieu of T2D, suggests that the factors associated with the diabetes in the MKR mice (e.g., insulin resistance, hyperglycemia) may cause a shift in the marrow osteoclast precursor pool or a phenotypic shift in the sensitivity of the precursors to osteoclast regulatory factors.
Despite a paucity of data, there has been general acceptance of the view that increased activity by osteoclasts does not play a role in T2D-related bone disease because of low bone turnover. Based on clinical biopsy bone formation data from a heterogeneous population (body mass index, sex and ancestry) Krakauer et al.
speculated that the metabolic effects of poor glycemic control leads to increased bone resorption, but then low bone turnover retards age-related bone loss [58
]. While such site-specific data is missing from the more recent clinical literature, biochemical data support the view that osteoclastic function is accelerated in non-obese males with T2D [59
]. Resolution of this issue in human subjects remains for future studies. Current data from the MKR mice, as well as prior studies of T2D rats [27
] also argue that alteration in osteoclast action may be present, though their contribution to the bone fragility in this disease remain unclear.
The biomechanical results from the current studies show that MKR bones were significantly weaker and less stiff than WT femurs. These changes are attributable to the smaller bone size and cross-sectional moment of inertia of the MKR bones. However, we also observed changes in the fracture resistance (ductility) of the MKR bones that suggest there is also a significant alteration in tissue quality of the diabetic bone. Specifically, the MKR femurs exhibited approximately 50% greater post-yield deflection (PYD) than WT. Compositionally, such changes can result from decreased mineral content, but this was not seen in the mineralization measurements of MKR bones. Alternatively, increases in bone collagen content or alterations in collagen cross-linking with other matrix constituents can also produce these changes in fracture behavior; however these could not be examined directly in the current studies because of the limits imposed by the other investigational techniques used.
Although osteoporotic fracture is more prevalent in women, the effects on men often result in greater morbidity and mortality, principally because related fractures occur later in life and males have a greater co-morbidity risk [60
]. In several clinical studies fracture risk associated with diabetes appears to differ between men and women; diabetes was associated independently with increasing risk of non-vertebral fractures among men but not women [9
]. Male diabetics are also more prone to be non-obese and the sexual dimorphism may be related to obesity and greater androgenecity in women with hyperglycemic and hyperinsulinemic conditions [9
]. Nonetheless, the increased male risk cannot be explained as an effect of BMD; both men and women with T2D had similar BMD compared to normals. In men, there is an increased risk associated with T2D of short duration that has not yet been attributed to reduced bone mass, quality or some other factor [2
]. For these reasons we choose to study male animals, as have many previous researchers, and all studies should be repeated in females [26
While our MKR mice are smaller than WT, both groups exhibited normal and similar weight gain from 3 to 16 weeks of age (MKR: 177%, WT: 173%). This is in contrast to the drug-induced diabetic animals that exhibit metabolic abnormalities characteristic of uncontrolled diabetes, including body weight gain [26
]. Previous work has established that tissue wet weights of organs expressing IGF-I receptors, namely, brain, lung, heart, liver, spleen, kidney, perigonadal white fat, and skeletal muscle are not different between MKR and WT mice, except for total skeletal muscle from birth to 5 weeks, and muscle mass differences between MKR and WT mice resolve; similar muscle weight and strength are seen when measured at 8 weeks [34
]. Thus, the MKR mice allow the separation of the hyperglycemic and hyperinsulinemic effects from increases in body weight and adiposity.
In summary, we presented a mouse model of T2D, which progresses from severe insulin resistance to frank diabetes. The early onset of the disease, during adolescence and puberty, lead to slender adult bones resulting in reduced strength and stiffness, and also changes in fracture resistance. The slender bones in diabetic mice resulted principally from increases in osteoclastic activity, increases that paralleled the progression to diabetes. Cortical and cancellous bone responded similarly, with loss of bone mass and trabecular architecture in concert with the progression to diabetes. These data suggest that T2D results in a complex array of alterations to skeletal morphology (resulting from net resorptive cellular activity) and tissue composition that may not be readily assessed (i.e., DEXA) or treated (i.e., anti-resorptive or bone anabolic strategies) using traditional measures. Whether control of the hyperglycemia and/or insulin resistance will resolve these skeletal issues awaits further research.