Bisphosphonate treatment of type I diabetic mice prevents early bone loss but accentuates suppression of bone formation

Abstract

Type I (T1) diabetes is an autoimmune and metabolic disease associated with bone loss. Previous studies demonstrate that T1-diabetes decreases osteoblast activity and viability. Bisphosphonate therapy, commonly used to treat osteoporosis, is demonstrated to inhibit osteoclast activity as well as osteoblast apoptosis. Therefore, we examined the effect of weekly alendronate treatments on T1-diabetes induced osteoblast apoptosis and bone loss. Bone TUNEL assays identified that alendronate therapy prevents the diabetes-induced osteoblast death observed during early stages of diabetes development. Consistent with this, alendronate treatment for 40 days was able to prevent diabetes-induced trabecular bone loss. Alendronate was also able to reduce marrow adiposity in both control diabetic mice compared to untreated mice. Mechanical testing indicated that 40 days of alendronate treatment increased bone stiffness but decreased the work required for fracture in T1-diabetic and alendronate treated mice. Of concern at this later time point, bone formation rate and osteoblast markers, which were already decreased in diabetic mice, were further suppressed in alendronate-treated diabetic mice. Taken together, our results suggest that short-term alendronate treatment can prevent T1-diabetes-induced bone loss in mice, possibly in part by inhibiting diabetes onset associated osteoblast death, while longer treatment enhanced bone density but at the cost of further suppressing bone formation in diabetic mice.

Figure 1. Alendronate suppresses early stage diabetes-induced osteoblast death

Mouse bones were examined during early stage diabetes development, 7 days post-diabetes induction with or without alendronate treatment. Bars represent the average value ± SE (n≥5 per group). A) Percentage of TUNEL positive stained osteoblasts in femur bone. Data was analyzed by 2-way ANOVA (charts on right) followed by Fisher post hoc analyses that determined significant differences * p< 0.05 (control vs diabetic), ** p<0.01 (untreated vs treated diabetic). B) Bax mRNA expression from control (white bars) and diabetic (black bars) non-treated and alendronate treated (+ Aln) mice. Bax expression is calculated relative to the housekeeping gene HPRT. Bars represent the average value ± SE (n≥5 per group). *denotes a significant difference (p<0.05) compared to control as determined by one-way ANOVA.

Figure 2. Long-term treatment with alendronate (40 days) prevents diabetes-induced trabecular bone loss

A) Representative microcomputed tomography isosurface images of trabecular bone volume/total volume (BV/TV) from distal femurs were obtained from control and diabetic mice treated with or without alendronate at 40 days post-diabetes induction. B) Average BV/TV and bone mineral density (BMD) in control (white bars) and diabetic (black bars), non-alendronate treated and alendronate treated mice. Bars represent the average value ± SE (n=7–8 per group). * p<0.05, ** p<0.001 as determined by one-way ANOVA.

Figure 3. Decreased cortical thickness in diabetic mice

Representative microcomputed tomography isosurface images of cortical thickness from the mid-diaphysis region of femurs were obtained from control and diabetic mice treated with or without alendronate. Graphical representation of cortical thickness in millimeters is displayed from control (white bars) and diabetic (black bars), non-alendronate treated and alendronate treated mice. Bars represent the average value ± SE (n=7–8 per group). * p<0.05 compared to respective control. ** p<0.05 compared to untreated diabetic bone as determined by one-way ANOVA.

Figure 4. Diabetes induces marrow adiposity and alendronate therapy decreases marrow adiposity

Representative images of bone marrow adiposity from distal femurs were obtained from control and diabetic mice treated with or without alendronate for 40 days. Graphical representations of adipocyte number per total marrow area and aP2 mRNA expression are displayed from control (white bars) and diabetic (black bars), non-alendronate treated and alendronate treated mice. Bars represent the average value ± SE (n=7–8 per group). RNA levels are calculated relative to the housekeeping gene HPRT. Data analyzed by one-way ANOVA, * p<0.05, **p<0.01.

Figure 5. Alendronate increases bone stiffness while diabetes and alendronate reduce force to fracture

Three-point bending tests were performed on femurs from control (white bars) and diabetic (black bars) non-treated and alendronate treated mice. Stiffness, ultimate stress, energy and work to fracture (area under the stress strain curve) were calculated and represented as average value ± SE (n≥7–8 per group). Work to fracture was analyzed by 2-way ANOVA to determine the effects of bisphosphonate treatment, diabetes and bisphosphonate treatment × diabetes on femur strength parameters. Fisher post hoc test was used to compare differences between groups. The rest of the graphs were analyzed by one-way ANOVA followed by Fisher post hoc tests to compare differences between groups, *p<0.05, **p<0.01.

Figure 6. Representative scanning electron microscopy (SEM) images of fracture surfaces of bones obtained from alendronate treated control and diabetic mice

After strength testing, the bone fracture surfaces were examined. Noted are the tensile (T) and compressive fracture areas (C). White arrows point to circumferential cracks that run parallel to the bone surface.

Figure 7. Alendronate effects on bone resorption markers

Serum TRAP5 concentration, cathepsin K mRNA levels, and osteoclast surface were analyzed in non-treated and alendronate treated in control (white bars) and diabetic (black bars) mice at the 40 day time point. Bars represent the average value ± SE (n=7–8 per group). Data was analyzed by one-way ANOVA followed by Fisher post hoc tests to compare differences between groups, *p<0.05, **p<0.01.

Figure 8. Bone formation is decreased at 40 day time point as a result of both diabetes and bisphosphonate therapy

A & B) Serum osteocalcin concentration and tibia mRNA osteocalcin expression was analyzed in non-treated and alendronate treated in control (white bars) and diabetic (black bars) mice. Bars represent the average value ± SE (n=7–8 per group). RNA levels are calculated relative to the housekeeping gene HPRT. C) Mice were injected with calcein dissolved in saline 7 and 2 days prior to harvest. L3–L4 vertebrae sections were photographed under UV light and the distance between the calcein lines were measured. Bone formation rate is graphed and bars represent the average value ± SE (n=7–8 per group). One-way ANOVA followed by Fishers post hoc tests determined differences among the groups. * p<0.05 compared to respective control, ** p<0.01, ***p<0.001.