gms | German Medical Science

Objectives: More than 6 million people in Germany are shift workers. Due to the disruption of the circadian clock, around 10 percent of them develop a syndrome called shift work disorder with cognitive impairments and metabolic alteration including an increased risk for diabetes. Although several studies showed that shift workers suffer from reduced bone density and increased incidence of fractures, the underlying mechanisms are poorly understood. Recently, we were able to show that global disruption of the central circadian transcription factor Bmal1 in mice impairs bone metabolism and fracture healing, however the transferability to human shift workers is limited. Whereas the global Bmal1 knock-out model depletes both the central pacemaker in the hypothalamus and the peripheral clocks intrinsically expressed in bone cells, we were looking for a more appropriate model to simulate bone metabolism in shift workers. Therefore, we investigated the bone structure of an advanced mouse model with brain-specific disruption of the circadian clock.

Methods: To deplete the circadian clock specifically in the hypothalamus, we used Synaptotagmin 10 – Cre (Syt-Cre) mice and mated them with Bmal1Flox mice. In this model, the Bmal1 gene stays functional in all peripheral tissues except testis, enabling rhythmic gene expression in bone cells. All investigated mice were homozygous for the Syt-Cre gene. Mice with a Bmal1 knock-out and a floxed allele (Bmal1 -/flox) were used as mutants (Bmal1^SCN) whereas littermates with a Bmal1 knock-out and a Bmal1 wild type allele (Bmal1 -/+) served as controls. Mice were kept under 12/12 hrs light/dark conditions followed by four weeks of constant darkness to assess the effect of the endogenous clock and to exclude any effects of exogenous light-dark cycles. Bone structures of femur, tibia and lumbar vertebrae 5 and 6 were analyzed with micro-CT and histology in 15 weeks old female mice.

Results and conclusion: Running-wheel activity in Bmal1^SCN mice was rhythmic under 12/12 hrs light/dark conditions. Under constant darkness, controls still showed rhythmic running-wheel activity, whereas Bmal1^SCN mice were behaviorally arrhythmic, confirming the disrupted circadian clock. Analyses of the cortical areas of femur and tibia of Bmal1^SCN mice revealed a reduced cortical thickness. Additionally, the trabecular structure of both bones was significantly altered with diminished bone volume to total volume and reduced trabecular numbers. Moreover, the trabecular architecture of the lumbar vertebrae 5 and 6 of Bmal1^SCN mice was significantly altered compared to control littermates. Hence, the corresponding changes in the bone morphology of Bmal1^SCN mice show that a disruption of the central circadian clock impairs bone metabolism and structure even in the situation of genetically intact circadian rhythms in bone cells. Therefore, this model holds great potential to explore bone loss in shift workers in order to prevent and treat osteoporosis.