Diabetes Stimulates Osteoclastogenesis by Acidosis-Induced Activation of Transient Receptor Potential Cation Channels

Abstract

Patients with type 1 diabetes have lower bone mineral density and higher risk of fractures. The role of osteoblasts in diabetes-related osteoporosis is well acknowledged whereas the role of osteoclasts (OCLs) is still unclear. We hypothesize that OCLs participate in pathological bone remodeling. We conducted studies in animals (streptozotocin-induced type 1 diabetic mice) and cellular models to investigate canonical and non-canonical mechanisms underlying excessive OCL activation. Diabetic mice show an increased number of active OCLs. In vitro studies demonstrate the involvement of acidosis in OCL activation and the implication of transient receptor potential cation channel subfamily V member 1 (TRPV1). In vivo studies confirm the establishment of local acidosis in the diabetic bone marrow (BM) as well as the ineffectiveness of insulin in correcting the pH variation and osteoclast activation. Conversely, treatment with TRPV1 receptor antagonists re-establishes a physiological OCL availability. These data suggest that diabetes causes local acidosis in the BM that in turn increases osteoclast activation through the modulation of TRPV1. The use of clinically available TRPV1 antagonists may provide a new means to combat bone problems associated with diabetes.

Figure 1. Diabetes induces osteoclast activation.

(a) Graph shows urine glucose levels in control and diabetic animals. (b) Representative microphotographs of TRAP^pos osteoclasts at 5 weeks of DM. Scale bar: 20 μm. (c) Graph shows the kinetics of osteoclast activation up to 11 weeks of DM. Data (mean ± SEM) express the number of TRAP^pos osteoclasts/mm of endosteal bone. (d) Graph shows the ratio between bone and total tissue area at 5 weeks of DM. Charts representing the levels of bone resorption product CTX-I (e) and osteocalcin (f) in PB plasma at 5 weeks of DM. *p < 0.05 Diabetics vs Controls; ^†p < 0.05 vs 1 and 3 weeks of DM. Studies were performed on 4 mice per group.

Figure 2. Effects of HG and hypoxia on osteoclast differentiation and activation in vitro.

Bar graphs (a) and representative microphotographs (b) show the abundance of TRAP^pos osteoclasts derived from BM-MNCs cultured under normal/high glucose and normoxic/hypoxic conditions. Bar graphs (c) and representative microphotographs (d) show the abundance of TRAP^pos osteoclasts derived from BM-MNCs exposed to the hypoxia mimic DMOG. Data are expressed as mean ± SEM. *p < 0.05 vs normoxia and **p < 0.01 vs 0 mM DMOG. n = 3 samples/group. Scale bar: 200 μm. Bar graphs show the level of TNFα in supernatants of BM-MNCs cultured in normoxia/hypoxia and normal/high glucose (e). n = 4 samples/group.

Figure 3

Role of acidosis in hypoxia-induced osteoclast activation: pH levels in media collected from BM-MNCs cultured under hypoxia or normoxia (a) or in the presence of buffering systems to prevent acidosis (c). Bar graph shows the abundance of TRAP^pos osteoclasts derived from BM-MNCs cultured under basal or acidosis conditions (b) or following addition of 2% CO₂ or HEPES to buffer hypoxia-induced acidosis (d). Bar graph shows the abundance of TRAP^pos osteoclasts derived from BM-MNCs of healthy or diabetic animals (e). Data are expressed as mean ± SEM and **p < 0.01 vs. normoxia and basal pH; *p < 0.05 vs. Basal pH controls, ***p < 0.001 vs Basal pH controls, ^†p < 0.05 vs pH 7.2 controls, ^††p < 0.05 vs Basal pH diabetes. n = 6 samples/group for A, 18 samples/group for (b), and 4 samples/group for (c–e).

Figure 4. Insulin replacement does not revert acidosis and osteoclastogenesis.

Graph shows pH of PB (a) and BM supernatants (b) from mice with STZ-induced DM given insulin replacement (grey bars) or vehicle (black bars) as compared with non-diabetic control mice (white bars). Graph shows the number of TRAP^pos osteoclasts/mm of endosteal bone (c). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. non-diabetic; n = 6 samples/group.

Figure 5. Expression of TRPV1 ion channel in diabetes.

Representative microphotographs displaying TRPV1 staining (green) in BM-derived osteoclasts in normoxic and hypoxic conditions. Nuclei are stained with dapi (blue). Scale bar: 50 μm. (a) Graph illustrates the increase of TRPV1 intensity in cells cultured in hypoxia as compared to normoxia. Data are expressed as the average of TRPV1 intensity per single cells, corrected for the background (b). Representative western blot and graph showing TRPV1 expression in BM-MNCs isolated from diabetic and control mice. Tubulin was used for normalization (c). Data are expressed as mean ± SEM. *p < 0.05 vs normoxia and non-diabetic controls; n = 4 samples/group.

Figure 6. Effects of TRPV1 agonists and antagonists on osteoclast differentiation.

Representative microphotographs (a,b) and bar graphs (c) show the abundance of TRAP^pos osteoclasts derived from BM-MNCs cultured with TRPV1 agonists (a) capsaicin (10 μM) or olvanil (10 μM) and antagonists (b) capsazepine (10 μM) or SB-366791 (10 μM) under normal (7.4) or low (7.2) pH. Data are expressed as mean ± SEM. *p < 0.05 and **p < 0.01 and ***p < 0.001 vs control at basal pH, ^†p < 0.05 and ^††p < 0.01 vs control acidosis. n = 7 samples/group. Scale bar: 200 μm.

Figure 7. TRPV1 antagonist SB-366791 prevents osteoclast formation in diabetic animals.

Representative microphotographs (a) and bar graph (d) show the abundance of TRAP^pos osteoclasts/mm of endosteal bone of controls and diabetic animals (5 weeks) treated with vehicle or TRPV1 antagonist SB-366791. Scale bar: 20 μm. Data are expressed as mean ± SEM. ***p < 0.001 vs vehicle controls. n = 6 animals/group.

Figure 8. Schematic representation showing the proposed mechanisms for the development of osteoporosis in the setting of DM1.

Type 1 diabetes induces hypoxia in the bone marrow, which determines local metabolic acidosis. The decrease in pH stimulates the expression and activation of the polymodal cation channel TRPV1 which, in turn, promotes the activation of osteoclasts, leading to osteoporosis. Therapies aimed at preventing TRPV1 stimulation (ie. by using the TRPV1 antagonist SB366791) would prevent the increased osteoclast activation observed in the setting of DM1, thus protecting the diabetic bone from osteoporosis.