Sex-specific characterization of a novel metabolic osteoporosis phenotype induced by NOS-inhibition and Western diet in Wistar rats

BACKGROUND: Multiple aspects of the metabolic syndrome are linked to impaired bone health, yet most animal models address only single contributing factors. Here, a male and female "two-hit" rat model of metabolic osteoporosis combining a high-fat/high-fructose "Western" diet (WD) and N(ω)-nitro-L-arginine-methyl-ester (L-NAME) is introduced and characterized. METHODS: Female Wistar rats were randomized into three groups: [i] Chow, [ii] Chow+L-NAME, and [iii] WD+L-NAME. Groups [i] and [iii] were replicated in males. After 16 weeks, femora were analyzed by micro-computed tomography, histology, and RNA-sequencing. Serum analyses included bone turnover markers and steroid hormones (LC/MS-MS). RESULTS: WD+L-NAME induced bone loss in both sexes, e.g. with the bone-volume-to-tissue-volume-ratio (BV/TV) being significantly reduced in females ([i] 53.8 ± 2.0, [iii] 43.0 ± 2.0; p < 0.01) and males ([i] 33.8 ± 7.4, [iii] 18.5 ± 9.4; p < 0.05). Female Chow+L-NAME rats exhibited elevated osteoclast numbers per bone surface ([i] 4.0 ± 1.8, [ii] 9.9 ± 3.6; p < 0.05), while no such changes occurred in WD+L-NAME females ([iii] 4.2 ± 2.7; n.s.). The Wnt-inhibitor sclerostin was significantly elevated in males on WD+L-NAME ([i] 278 ± 133, [iii] 931 ± 454 pg/ml; p < 0.05). While steroid hormones were not significantly altered, RNA-sequencing revealed a downregulation of bone formation-related pathways in both sexes under WD+L-NAME, with extracellular matrix-related gene terms being primarily affected by WD. CONCLUSION: The combination of WD+L-NAME induces a metabolic osteoporosis phenotype in male and female rats. It is thereby representing the first "two-hit" model for metabolic osteoporosis with a phenotype in both sexes. Elevated DKK1 and sclerostin in males suggest Wnt-signalling involvement, while WD predominantly impacts extracellular matrix regulation.