Early Trabecular Compensation in Femoral Bone of the Diabetic NOD*B10 foz/foz Mouse

Abstract

Bone mineral density (BMD) does not adequately predict bone fracture risk in contemporary type 2 diabetic patients. The rising prevalence of younger onset diabetes and obesity together reveal an endemic fragility for diabetic patients with equal or higher BMD than non-diabetics. Other factors contribute to this paradoxical fracture risk jeopardising skeletal acquisition and lifetime bone remodelling capacity.

NOD*B10 mice are susceptible to obesity and diabetes unlike the BALB/c strain which are only obese under identical genetic (foz/foz) stress. These strains enable investigation of diabetes-specific features distinct from obesity in the co-morbid NOD*B10 group. High fat (HF) feeding accelerates disease acquisition, enabling investigation of trabecular bone parameters and gene expression changes of the femur with earlier diabetes onset. We hypothesised that younger diabetic mouse bone would corroborate the BMD paradox, but expected structural and biochemical changes in trabecular bone from enhanced metabolic adaptation in this growth-emphasis project.

Female wild type and foz/foz littermates were raised and fed a standard or HF diet from 4 to 13 weeks of age on either BALB/c (n=29) or NOD*B10 (n=30) background. Fed-state body weight, insulin and blood glucose concentrations were obtained at end point. Serum was collected at sacrifice and hind limbs were dissected. Right femurs were preserved for micro-CT scanning and BMD measurement. Left femurs were processed for osteogenic gene expression profile by PCR array. ELISA was performed on serum samples to determine peripheral concentrations of a selected protein implicated by the PCR array.

At 13 weeks of age, HF-fed NOD*B10 foz/foz mice were diabetic and obese, whereas corresponding BALB/c mice were merely obese. Healthy mice and diet or genetic stress groups achieved desired phenotype spectra for comparison. HF-foz/foz NOD*B10 mice revealed equivalent BMD to non-diabetic groups as expected.

Diabetic NOD*B10 mice reflected thinner trabeculae than HF-WT mice, and a tendency (albeit statistically insignificant) for more trabeculae implicating expanded surface area within the distal femur. Mice of a given strain were otherwise microstructurally alike, with consistent cortical indices, bone volume fraction and BMD. Unexpectedly inter-strain comparisons were not possible by the stark contrast in trabecular features between NOD*B10 and BALB/c mice, irrespective of treatment conditions.

Despite largely maintained femoral microstructure amongst NOD*B10 littermates, HF-foz/foz mice revealed a quiescent osteogenic profile with under-expression of Runx2, Igf1r, Fgfr2 and Phex insinuating early osteoblast inhibition and subsequent mineralisation loss. Chow-foz/foz mice revealed relative over-expression of Fgf1, Anxa5 growth factor and mineralisation genes suggesting metabolic stimulation with establishing pre-diabetes. This distinction prompted analysis of FGF1 protein expression in serum. Difficulties in the result determination proved inconclusive, however putatively suggested raised FGF1 levels in diabetic serum.

Taken together, these findings support the clinical notion of trabecular bone appraisal with BMD measurement. Earlier diabetes onset provokes a younger and altered mode of skeletal dysregulation to conventional aging studies. This project characterises the femoral bone microstructure of the female NOD*B10 foz/foz model for the first time, demonstrating inherent differences to the BALB/c strain which are vital to future study design and interpretation. Show more