Risk factors for fragility fractures and role of Wnt signaling in type-1 diabetes related bone fragility: clinical and preclinical studies

BACKGROUND Patients with Type 1 diabetes (T1D) have low bone mass and up to five times higher risk of hip fracture than normal peers, leading to an increased morbidity and mortality. However, risk factors for fractures in T1D have not been clearly identified and fractures are still a poorly screened diabetic complication. In a preliminary clinical study, we found high circulating levels of the Wnt inhibitor sclerostin in patients with T1D relative to normal controls. This result would be consistent with findings of decreased serum markers of bone formation in humans and preclinical models of T1D, suggesting reduced bone turnover in T1D. However, diabetes-related mechanisms underlying a lower bone quality and increased risk of fractures in diabetics are, similarly to clinical factors, still not well established. The overall objectives of the study were to evaluate risk factors for fractures in T1D and mechanisms underlying bone fragility in an experimental model of T1D. STUDY 1: CLINICAL STUDY OBJECTIVE: To determine clinical diabetes-related risk factors for fractures in type 1 diabetes. RESEARCH DESIGN AND METHODS: History of bone fragility fractures occurring after T1D diagnosis was assessed by questionnaire in this cross-sectional study in 600 T1D subjects. Glycated hemoglobin A1c (HbA1c) over the previous 5 years was used as an index of long-term glycemic control; complications were adjudicated by physician assessment. Multinomial logistic regression models were used to assess the associations between diabetes-related risk factors and fracture history. RESULTS: One-hundred-eleven patients (18,5%) reported at least one fracture; of these 73.8% had only one and 26.2% had more than one fracture. Average age was 41.9 ± 12.8 years, with even gender distribution; disease duration was 19.9 ± 12.0 years; and BMI was 24.4 ± 3.7 kg/m2. The 5-year average HbA1c was 7.6 ± 1.0% (60 mmol/mol). In adjusted models, reduced risk for 1 fracture was found in those with higher eGFR (RRR 0.22 [95%CI: 0.06-0.83] for 1 unit increase in lneGFR, p = 0.03) and increased risk in those with neuropathy (RRR 2.57 [1.21-5.46], p = 0.01). Increased risk for >=2 fractures was found in subjects in the highest tertile of HbA1c (>=7.9%) compared with the lowest tertile (<=7.17%) (RRR 3.50 [1.04-11.7], p = 0.04) and of disease duration (>=26 years versus <14 years) (RRR 7.59 [1.60-36.0], p = 0.01). Summary study 1: Poor glycemic control and long exposure to the disease are independent diabetes-related risk factors for multiple bone fractures in type 1 diabetes. STUDY 2: PRECLINICAL STUDY OBJECTIVE: To test the potential role of sclerostin and Wnt signaling in diabetes induced bone disease. RESEARCH DESIGN AND METHODS: We introduced the sclerostin-resistant Lrp5A214V mutation, associated with high bone mass (HBM), in mice carrying the Ins2Akita mutation (Akita), which causes hyperglycemia and hypoinsulinemia within 5 weeks after birth. Bone microarchitecture and body composition were longitudinally evaluated respectively by in vivo µ-CT and DXA. Glucose metabolism was evaluated by random blood capillary measurements and intraperitoneal glucose and insulin tolerance tests. RESULTS: Bone mass by DXA was significantly higher in Akita/HBM relative to Akita littermates at 12 weeks (88.2 ± 5.2 vs 67.9 ± 4 mg/cm2; p <0.001; n = 7-11), persisting higher for up to 26 weeks (90.2 ± 3.0 vs 70.4 ± 1.7mg/cm2; p <0.001 n = 5) despite overt diabetes. Further analysis by µCT at age 20 weeks revealed lower trabecular bone volume/total volume (BV/TV) in Akita compared to wild type (WT) mice (0.2 ± 0.02 vs 0.35 ± 0.05; p <0.05; n = 3-5). Conversely, both trabecular (Tb) and cortical (Ct) parameters were significantly higher in Akita/HBM mutants compared to Akita littermates, including total Ct area (1.6 ± 0.06 vs 1.2 ± 0.07mm2), bone area (0.9 ± 0.1 vs 0.6 ± 0.05mm2), and Ct thickness (0.2 ± 0.02 vs 0.1 ± 0.01 mm, p <0.001, n = 5-7). Tb BV/TV and Tb thickness were also higher in Akita/HBM mutants relative to Akita littermates (0.4 ± 0.05 vs 0.2 ± 0.02; and 0.1 ± 0.02 vs 0.09 ± 0.03mm, respectively, p <0.001, n = 3-5). We found no significant differences in total Ct area between Akita and WT mice, consistent with observations in humans with T1D. As expected, both Akita and Akita/HBM mutants developed diabetes (non-fasting blood glucose >300 mg/dl), albeit with different onset timing. At 8 weeks, only 40% of Akita/HBM mice had developed hyperglycemia, compared to 90% of Akita mice (n = 10). Only at 12 weeks were most Akita/HBM mice hyperglycemic. Intriguingly at age 6 and 8 weeks, glucose tolerance was significantly better in Akita/HBM relative to Akita mice (p <0.05 for difference in areas under the curve, AUC; n = 3-6). Likewise, insulin sensitivity (by intraperitoneal insulin tolerance test) was higher in the Akita/HBM compared to the Akita group (p <0.01 for difference in AUC; n = 4-8) at age 7 weeks. Summary study 2: The metabolic changes caused by hypoinsulinemia (chronic hyperglycemia) do not alter the consequence of sclerostin resistance and Wnt hyperactivation on bone. Furthermore, Wnt activation retards the onset of metabolic abnormalities in T1D. CONCLUSIONS Our data clearly show that T1D patients should be carefully screened for fragility fractures and we propose risk factors for any and multiple fragility fractures to use for a model of prediction for fractures. We have proved that HbA1c, disease duration, presence of neuropathy and eGFR values could be targeted for prevention of fractures in diabetes. With our preclinical study we explored for the first time in vivo the effect of T1D and sclerostin resistance on bone, demonstrating that targeting Wnt signaling protects bone mass in T1D. We also potentially target a role of sclerostin resistance to improve not only bone fragility but also glucose metabolism. This study provides novel scientific inputs in the relationship between glucose homeostasis and bone health and may offer new avenues for assessment and treatment of bone alterations in diabetes.