Leptin and the sympathetic nervous system have a unique role in linking nutritional status to skeletal metabolism in mammals. Such a regulatory mechanism has not been identified in birds but would be beneficial to signal information about energy reserves to an organ system essential for locomotion, reproduction, and survival. To explore this potential role of leptin and the sympathetic nervous system in birds, an ex vivo chick tibiotarsal model was used to test the effects of leptin and sympathetic activity on longitudinal bone growth and the expression of chondrocyte markers. Reverse transcription-PCR analysis revealed the expression of chicken leptin receptor mRNA as well as both α-adrenergic (α-1A, α-2A, α-2B, α-2C) and β- adrenergic (β-1, β-2) receptor subtype mRNA in the whole bone. Incubation with norepinephrine (NE;- 0, 10, or 100 μ-M for 4 d) caused a significant increase in distal condyle length as compared with vehicle-treated, contralateral tibiotarsi. In contrast, no change in condyle length was detected after leptin treatment (0 or 10 nM or 1 μ-M for 4 d). Analysis of cell proliferation by bromodeoxyuridine incorporation revealed no increase in bromodeoxyuridine-positive cells in the condyles in response to leptin or NE treatments. Real-time PCR analysis showed that NE enhanced type X collagen mRNA expression, a marker of mature hypertrophic chondrocytes, with no effect on type II collagen mRNA, the matrix protein secreted by proliferating chondrocytes. Leptin treatment had no effect on the expression of either matrix protein. Treatment with agonists specific for α- or β-adrenergic receptors indicates that the activation of α-adrenergic receptors is most likely responsible for the sympathetic effect on type X collagen gene expression. These results suggest that NE and other sympathetic agonists have positive effects on bone elongation and the changes in critical genes associated with this process. These neurotransmitters may facilitate this by promoting chondrocyte maturation. These studies represent novel evidence suggesting a role of sympathetic tone in the regulation of skeletal growth in avian species.
Bibliographical noteFunding Information:
We thank Marion Zillhardt, Audrey Mayer, Ngozika Okoye, and Courtney Blohm (University of Minnesota) for excellent technical assistance. Kent Reed (University of Minnesota) generously donated turkey heart RNA for the initial verification of PCR primer sets used for amplification of the turkey and chicken β1-adrenergic receptor genes. Cathy Carlson and Ann Undersander (University of Minnesota) provided invaluable advice on bone histopreparation and immunostaining. This research was supported by funds from the Minnesota Agricultural Experiment Station (to LJM: MIN-16-016).
- Adrenergic receptor