Bone's responses to mechanical loading are impaired in type 1 diabetes

Abstract

Diabetes adversely impacts many organ systems including the skeleton. Clinical trials have revealed a startling elevation in fracture risk in diabetic patients. Bone fractures can be life threatening: nearly 1 in 6 hip fracture patients die within one year. Because physical exercise is proven to improve bone properties and reduce fracture risk in non-diabetic subjects, we tested its efficacy in type 1 diabetes. We hypothesized that diabetic bone's response to anabolic mechanical loading would be attenuated, partially due to impaired mechanosensing of osteocytes under hyperglycemia. Heterozygous C57BL/6-Ins2(Akita)/J (Akita) male and female diabetic mice and their age- and gender-matched wild-type (WT) C57BL/6J controls (7-month-old, N=5-7 mice/group) were subjected to unilateral axial ulnar loading with a peak strain of 3500 με at 2 Hz and 3 min/day for 5 days. The Akita female mice, which exhibited a relatively normal body weight and a mild 40% elevation of blood glucose level, responded with increased bone formation (+6.5% in Ct.B.Ar, and 4 to 36-fold increase in Ec.BFR/BS and Ps.BFR/BS), and the loading effects, in terms of changes of static and dynamic indices, did not differ between Akita and WT females (p ≥ 0.1). However, loading-induced anabolic effects were greatly diminished in Akita males, which exhibited reduced body weight, severe hyperglycemia (+230%), diminished bone formation (ΔCt.B.Ar: 0.003 vs. 0.030 mm(2), p=0.005), and suppressed periosteal bone appositions (ΔPs.BFR/BS, p=0.02). Hyperglycemia (25 mM glucose) was further found to impair the flow-induced intracellular calcium signaling in MLO-Y4 osteocytes, and significantly inhibited the flow-induced downstream responses including reduction in apoptosis and sRANKL secretion and PGE2 release. These results, along with previous findings showing adverse effects of hyperglycemia on osteoblasts and mesenchymal stem cells, suggest that failure to maintain normal glucose levels may impair bone's responses to mechanical loading in diabetics.

Keywords: Akita diabetic mouse; Bone formation; Fluid flow; Osteocyte mechanosensitivity; Ulnar mechanical loading.

Fig. 1

(A) In vivo ulnar loading model. Peak strain was validated using strain gauges placed on the relative flat area (1-2mm proximal of the mid-shaft). Induced bone formation was evaluated in the cross-sections of the mid-shaft (dotted lines). (B) Calibrated peak load magnitudes to achieve similar 3500 με surface strain in the four experimental groups.

Fig. 2

Intracellular calcium imaging. (A) Osteocytes were exposed to fluid flow stimulation in a laminar flow chamber. (B) Osteocytes were dyed with Fluo-4 and imaged for 10 min. (C) A representative calcium response and the quantified spatiotemporal parameters (number of peaks, 1^st peak magnitude (m₁), 1^st peak response time (t₁) and relaxation time (t₂)).

Fig. 3

Load-induced changes in bone area. (A) Females Akita with mild hyperglycemia showed similar bone formation as WT; (B) The effect of loading was completely abolished in Akita males with severe hyperglycemia. The p values were for Mann-Whitney U tests. Data are presented as mean ±SD.

Fig. 4

Representative cross-section images showing bone labels for (A) normal and Akita females and (B) normal and Akita males. Bar=0.2mm.

Fig. 5

Hyperglycemia impaired osteocytes’ multiple [Ca²⁺]_i responses to fluid flow. (A) No significance among three groups on the fraction of cells responding with one peak or more; (B) Significant decrease in the fraction of cells responding with multiple peaks in the hyperglycemic medium (35%) versus regular (51%) and osmotic control media (50%). (C) The average peak number decreased in media with elevated osmolarity. (D) No difference in the 1^st peak magnitude. (E) Faster responding time from the onset of flow to the 1^st peak in the osmotic control medium. (F) No difference in the relaxation time from the 1^st peak to baseline. Regular medium contained 5.5mM D-glucose, while the osmotic control medium and hyperglycemic medium contained additional 20mM L-glucose, and 20mM D-glucose, respectively. The flow tests were repeated 6-8 times with 444-640 cells analyzed per medium condition. * p<0.05. Bars indicate one standard deviation.

Fig. 6

Hyperglycemia impaired osteocyte's downstream responses to fluid flow. (A) Caspase 3/7 expression; (B) Soluble RANKL expression; (C) PGE2 concentration in media. Data were normalized to those under static no-flow condition. All tests were repeated 4 times. * indicated p<0.05 vs. hyperglycemic condition (ANOVA with post hoc Tukey tests). Bars indicate one standard deviation.