Sustained Morphine Delivery Suppresses Bone Formation and Alters Metabolic and Circulating miRNA Profiles in Mice
Authors: Carvalho Adriana Lelis , Brooks Daniel J , Barlow Deborah , Houseknecht Karen , Bouxsein Mary , Lian Jane B , King Tamara , Farina Nicholas H , Motyl Katherine J 
Published: May 2021

Abstract
Opioid use is associated with a three to four-fold increase in fracture risk, which is higher in males compared to females. Although one component of that risk may be falls, there is also evidence of altered bone remodeling. However, the mechanisms of these effects are not entirely clear. Our aim was to develop a mouse model of opioid-induced bone loss and study the impact on bone turnover and potential miRNA-mediated regulatory mechanisms. We evaluated the effects of sustained morphine treatment on the skeleton and metabolism of male and female C57BL/6J mice by treating with vehicle (0.9% saline) (n=9–11) or morphine (18 mg/kg) (n=11) using subcutaneous osmotic minipumps for 25 days. Morphine did not influence body weight or food intake, but did reduce fat mass in both sexes. All mice treated with morphine had higher resting energy expenditure and respiratory quotient, indicating a shift toward carbohydrate metabolism. After 25 days of treatment, microComputed Tomography (µCT) analyses indicated that morphine-treated male mice lost 15% of trabecular bone volume fraction (Tb.BV/TV) and 14% of Tb. bone mineral density (BMD) (p&lt;0.05) in the distal femur compared to vehicle, but there were no changes in cortical bone. Females did not lose bone, suggesting differences may be hormone-related. Despite these sex differences, males and females had similar levels of morphine exposure, measured by LC-MS/MS. Histomorphometric analyses demonstrated that in males, morphine reduced bone formation rate (p&lt;0.05) compared to vehicle, but did not impact osteoclast parameters. Consistent with this, morphine reduced bone formation marker gene expression in the tibia of males (including Bglap and Dmp1). Circulating miRNA profiles were interrogated in serum collected at day 12 from vehicle- and morphine-treated male and female mice. While very few changes were present in females, there were 14 differentially expressed miRNAs in males treated with morphine that reached a threshold of ≥ 2-fold change and p&lt;0.01. After target pathway analysis (DIANA miR Path V.3 using experimentally validated miRNA/mRNA targets (Tarbase V.7)), we found that the four upregulated miRNAs (miR-484, -223-3p, -328-3p, and -3107-3p) were associated with 13 enriched KEGG pathways (p&lt;0.05), including fatty acid metabolism pathways (p&lt;0.001). Fatty acid metabolism has recently been linked to osteoblast function, and suppression of such pathways is consistent with the finding of increased respiratory quotient. In summary, morphine leads to trabecular bone loss due to reduced bone formation in males. miRNA findings indicate this may be due to altered metabolic control of mineralization. Further investigation into hormonal and metabolic dependency of morphine-induced bone formation changes could lead to clinical mitigation strategies for preventing the adverse effects of opioids on bone health.