Glicometabolic effects on bone metabolism: from new and different pathways to new diagnostic and therapeutic aspects

Background Diabetes and bone fragility are rapidly growing diseases, as osteoporosis could be defined as a complication of type 2 diabetes (T2D) (Epstein et al. 2016). Aging of populations worldwide will be responsible for an increased risk in the incidence of these two diseases. There is a pathophysiological link between the high fracture incidence and diabetes if compared with the non-diabetic state, has recently been recognized but not totally clear and understood. In fact, several mechanisms are involved in bone homeostasis by impairing the function of bone cells as osteocytes, as osteoblasts and osteoclasts, and/or modifying the structural characteristics of the bone tissue (Jiang and Xia 2018; Epstein et al. 2016). Furthermore, as osteoblasts and adipocytes both derived from the mesenchymal stem cell (MSC), their physiological differentiation could be modulated by several interacting pathways on which diabetes may shows its influence and disruption. Finally, is well known the alteration of different organs and systems during diabetic disease, involved in bone metabolism, as kidney, gut or vitamin D pathway. In fact, In the last few years, studies are focusing not only to clarify old pathways but also to discover new pathways among diabetes, obesity and bone metabolism, as Wnt signaling and the role of sclerostin, as irisin, as different formulations of vitamin D like calcidiol. Moreover, some complications derived from diabetes, as cardiac neuropathy, were not yet fully evaluated on their link to the axis diabetes-bone, leading a gap of knowledge to fill (Epstein et al. 2016; Napoli, Strollo, et al. 2014). So, many questions remain regarding the underlying mechanisms for greater bone fragility in diabetic patients and the best approach to risk assessment and treatment to prevent fractures. Specific aims 1) to evaluate the role of sclerostin in patients with type 2 diabetes and patients with LADA. 2) to investigate the relationship between irisin and body composition in subjects with osteoporosis and the impact of irisin levels on fragility vertebral fractures. 3) to evaluate the role of calcidiol on metabolic parameters and ß-cell function in subjects with impaired glycaemic control and insufficient vitamin D levels. 4) to evaluate the relationship between cardiac autonomic neuropathy and BMD in patients with diabetes. Materials and Methods 1) This cross-sectional study included 98 T2D and 89 LADA patients from the Action LADA and NIRAD cohorts. Patients were further divided according to MetS status. Non-diabetic subjects (n=53) were used as controls. Serum sclerostin, bone formation (P1NP) and bone resorption (CTX) were analyzed. 2) In this cross-sectional study, 36 overweight subjects affected by at least one vertebral osteoporotic fracture confirmed by a X-ray vertebral morphometry and 36 overweight non-osteoporotic subjects were enrolled. Serum irisin levels were measured using an irisin competitive ELISA. We evaluated lumbar spine and hip BMD and body composition using dual energy X-ray absorptiometry. To measure and monitor daily physical activity, each subject wore an armband for approximately 72 hours. 3) It is a double-blind placebo-controlled clinical trial enrolling subjects with IGT, IFG and T2D (20) and 25(OH)D <20 ng/ml. In this study were enrolled a total of 150 subjects and followed up for 6 months. Subjects were either assigned (50 per group) to 1) daily supplementation of 50 mcg of calcidiol (Arm A); 2) 25 mcg of calcidiol (arm B); 3) placebo (Arm C). Fasting blood glucose and Oral Glucose Tolerance Test (OGTT), HbA1c, 25 (OH) D, calcium, phosphorus, PTH, calciuria, phosphaturia, total cholesterol, HDL cholesterol and triglycerides were measured with laboratory kits used in clinical settings. Measurements of Ox-LDL, Hs-CRP, TNF-α, IL-6, esRAGE, sRAGE were performed at the laboratory. To evaluate insulin resistance were used the ISOGTT index and the evaluation of the model of insulin-resistance homeostasis (HOMA-IR). Beta-cell function was evaluated using the insulin secretion sensitivity index-2 (ISSI-2) 4) Fourty-nine people with T2D were enrolled. Tests to determine heart rate response to deep-breathing (expiratory-to-inspiratory ratio), heart rate response to lying-to-stand test (30:15 ratio) and blood pressure response to standing were performed to detect cardiac autonomic neuropathy, and dual energy X-ray absorptiometry scan of both the lumbar spine and femoral neck were performed to evaluate bone mineral density. Results 1) T2D subjects had higher sclerostin than LADA (p=0.0008, adjusted for sex and BMI), even when analysis was restricted to MetS subjects (adjusted p=0.03). Analyzing T2D and LADA separately, sclerostin was similar between subjects with and without MetS. However, a positive trend between sclerostin and number of MetS features was seen in T2D (p for trend=0.001) but not in LADA. Subjects with either T2D or LADA had lower CTX than controls (p=0.0003), and not significantly reduced P1NP. Sclerostin was unrelated to age or HbA1c but correlated with BMI (ρ=0.29; p=0.0001), HDL (ρ=-0.23; p=0.003), triglycerides (ρ=0.19; p=0.002) and time since diagnosis (ρ=0.32, p<0.0001). 2) No significant correlations were found between irisin and BMD at any site and between irisin with either lean or fat mass. Serum levels of irisin were not correlated with the daily physical activity. Serum irisin levels were lower in subjects with previous osteoporotic fractures than in controls (p= 0.032) and the difference in irisin levels remained significant after adjustment for creatinine (p=0.037), vitamin D (p=0.046), lean mass (p=0.02), lumbar BMD (p=0.023) and femoral BMD (p=0.032). 3) At baseline, subjects were (mean±SD) 63.8± 2.1 y.o., BMI was 27.4±1.2 kg/m2; serum glucose 115.1±8.4 mg/dL, HbA1c 6.4±0.6%, 25OHD 16.3±2.5 ng/mL. There were significant associations of 25OHD with ISOGTT (β=0.35; 95% CI, 0.14, 0.46) and β-cell function (ISSI-2; β = 0.15; 95% CI, 0.02, 0.28). At six months, 25OHD increased up to 48±3 ng/mL in Arm A (P<0.01) and to 36±5 ng/mL in Arm B (P<0.01); no significant changes in the Arm C. Subjects in Arm A had a lower risk of dysglycemia (HR= 0.85, 95% CI, 0.75-0.97 per SD increase) while no significant effects were observed in the Arms B or C. Both ISOGTT and ISSI-2 were improved in Arm A (P<0.05) while no significant changes were observed in Arm B or placebo. Serum levels of sRAGE decreased in Arm A [median 1354 (1069-1680) pg/ml (P<0.01), as compared with levels at study entry, but not in Arms B or C. No significant differences were observed for hsCRP, IL6, TNFα or lipid panel. 4) We analyzed preliminary results among two evaluations of BMD and CAN, not finding any significative difference. In fact, subjects with no CAN showed a normal BMD in 35,7% while the remaining part had osteopenia or osteoporosis (64,3%: osteopenia: 60,7%; osteoporosis: 3,6%). Evaluating BMD in subjects with CAN, 48,1 had a normal BMD while 51,9 had osteopenia (37%) or osteoporosis (14,8%). Conclusions 1) LADA patients present lower bone resorption compared to controls, similarly to T2D. Sclerostin is increased in T2D but not in LADA suggesting possible roles on bone metabolism in T2D only. 2) The data confirm an inverse correlation between irisin levels and vertebral fragility fractures, but no significant correlation was found with BMD or lean mass. Irisin may play a protective role on bone health independent of BMD. 3) Our findings indicate that high doses of calcidiol improved indices of glucose homeostasis in prediabetic subjects and decreased circulating sRAGE levels, suggesting a positive effect also on oxidative stress. 4) No significant correlations were found evaluating BMD in subjects with T2D and CAN.