The Relationship of Leptin and Leptin Bioavailability with Body Composition, Bone Quality and Osteoblast Functions in Adolescent Idiopathic Scoliosis

Introduction: Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional structural deformity of the spine and its etiology remains unknown. It mainly occurs in girls between 11 to 14 years old with a prevalence rate of 4%. This common spinal deformity can be associated with significant cosmetic, psychological, and clinical morbidities in severe cases. Current treatments for AIS are unsatisfactory, mainly because they are merely treating the phenotype of the spinal deformity but not the underlying etiology. Therefore, it is crucial to understand the etiopathogenesis of AIS so that effective therapeutic and preventive measures can be devised.;Previous studies have reported the association between AIS and low body weight, tall stature, increased arm span, low body mass index, delayed onset of menarche and low bone mass, which indicated abnormal systemic growth in patients with AIS. Leptin has been postulated as one of the etiologic factors of AIS because of its important physiological functions in neuro-osseous development affecting skeletal growth, the onset of puberty, energy expenditure and body composition. A previous study had reported lower circulating leptin in AIS girls than age- and sex-matched controls. However, the strength of leptin signal is not only determined by the leptin level, it could also be affected by soluble leptin receptor (sOB-R), its binding protein in circulation. sOB-R could modulate the serum leptin level, and affect the bioavailability of free leptin, the biologically active form, to its target tissues. Free leptin index (FLI) was developed to aid interpretation of data and provide an estimation of free leptin level.;The effects of leptin on bone metabolism are exerted indirectly through the central nervous system and directly on the peripheral tissues. Leptin was shown to have a catabolic effect on bone formation through the hypothalamus and the sympathetic nervous system (SNS). While the peripheral effects of leptin was shown to be anabolic to bone formation, promoting the proliferation of osteoblasts and chondrocytes, stimulating osteoblastic differentiation, mineralization, and inhibiting osteoclastogenesis and osteoclast activity. With regards to AIS, a previous study on bone marrow derived mesenchymal stem cells (MSCs) showed lower responses to leptin, and lower leptin receptor expressions in adipogenic and osteogenic MSCs. This study provided initial evidence that leptin related pathways might be abnormal in the bone cells of AIS patients.;This study aimed to: 1) Investigate if there is any abnormality in free leptin bioavailability by measuring the serum total leptin, sOB-R levels, and FLI in AIS and control subjects, and if it is associated with abnormal anthropometric parameters in AIS. 2) Investigate if free leptin bioavailability in AIS is associated with abnormal body composition by studying the difference in body composition and its correlations with serum total leptin, sOB-R, and FLI between AIS and control subjects. 3) Investigate if free leptin bioavailability in AIS is associated with abnormal bone quality by studying the difference in bone quality as measured by high resolution pQCT and its correlations with serum total leptin, sOB-R, and FLI between AIS and control subjects. 4) Investigate if there are abnormal functional responses to leptin and abnormal expression of leptin receptor in AIS by determining the effects of leptin on proliferation, differentiation, and mineralization of osteoblasts; and the expression levels of leptin receptor in osteoblasts isolated from intra-operative bone biopsies of AIS and control subjects.;Methods: 1) The study on free leptin bioavailability and anthropometric parameters included 207 AIS girls and 155 age- and gender-matched normal controls. Subjects with body mass index (BMI)>23.0 were excluded to avoid bias from the relatively high serum leptin level in overweight / obese subjects. Assessments included anthropometric and sexual maturity measurements: body weight, height, arm span, sitting height, BMI, and Tanner stages. Serum total leptin and sOB-R levels were measured with ELISA, and free leptin index (FLI) was calculated as the ratio of serum total leptin / sOB-R. 2) The study on free leptin bioavailability and body composition included 148 AIS girls and 116 normal controls, while anthropometric and sexual maturity parameters included 207 AIS girls and 155 normal controls. Body composition was assessed with bioelectrical impedance analysis (BIA). 3) The study on free leptin bioavailability and bone quality parameters included 207 AIS girls and 155 normal controls. Bone quality on the non-dominant distal radius was assessed with high resolution pQCT (HR-pQCT). In vivo bone strength was assessed with finite element analysis (FEA). 4) The study on functional responses and leptin receptor expression in osteoblasts included 12 AIS girls undergoing corrective spinal surgery and 6 control subjects undergoing unrelated surgery. Primary osteoblasts were isolated from intra-operative bone biopsies, and were cultured and assessed for the effects of leptin (0, 10, 100, 1000 ng/mL) on cell proliferation, differentiation, and mineralization by MTT assay, alkaline phosphatase activity assay and osteocalcin ELISA, and von Kossa staining respectively. Protein expressions of leptin receptor (LEP-R) under basal and osteogenic conditions were compared between AIS and controls by Western blot.;Results: 1) In the study on free leptin bioavailability and anthropometric parameters, AIS girls had higher sOB-R and lower FLI after adjusting for age and body weight, and these were associated with lower body weight and lower BMI. Significant correlation between serum total leptin and sOB-R was found in controls, but no correlation was observed in AIS girls. AIS girls also showed markedly weaker correlations between serum total leptin, FLI, and anthropometric parameters. 2) In the study on free leptin bioavailability and body composition, AIS girls had lower skeletal muscle mass, lower body fat, and percentage body fat, as well as weaker correlations between serum total leptin, FLI, and all body composition parameters. 3) In the study on free leptin bioavailability and bone quality, AIS girls had lower cortical volumetric bone mineral density (vBMD), lower trabecular number, higher trabecular separation, and higher structural model index (SMI) value. In correlation analysis with serum total leptin and FLI, AIS girls had distinctive correlation pattern with trabecular bone parameters that was not observed in the normal controls. 4) In the study on functional responses and leptin receptor expression in osteoblasts, control group showed increasing MTT signals to leptin in a dose dependent manner, while AIS group showed no proliferative response to leptin. For differentiation, control group showed strong and significant trend in ALP activity to increasing leptin concentrations in both day 6 and 14, but this trend was not observed in the AIS group. Osteoblasts from the control group secreted osteocalcin in an increasing dose dependent manner to leptin, but AIS group showed weak responses to leptin. For mineralization, the control group showed a mild increasing trend to increasing leptin concentrations, and again no trend was observed in the AIS group. No significant differences in the expression of leptin receptor under basal and osteogenic conditions were found between AIS and control group.;Discussion: This is the first study on the relationship of leptin bioavailability with body composition and bone quality in AIS. The results in this study suggested that abnormal free leptin bioavailability might exist in AIS girls and could lead to abnormal phenotypes such as lower BMI, abnormal body composition, and deranged bone quality that often manifested in AIS simultaneously. The importance of low bone mass in AIS and the presence of correlations between trabecular bone parameters and serum total leptin and FLI which were not observed in controls, have led us to further investigate the effects of leptin on primary osteoblasts in AIS. The osteoblasts isolated from AIS girls had no or very low response to leptin when compared with controls, which was shown to be independent of the difference in leptin receptor expression levels. This lack of response might be due to dysfunction of leptin signaling pathway, which might include structural or binding abnormalities in the leptin receptor or downstream signal molecules. This is an important finding and might serve to explain the low bone mass and deranged bone quality that is associated with AIS. This study provided new insights and new research directions on the etiopathogenesis of AIS. Future research could include studies on the downstream signal molecules along the leptin pathways that might be affected in AIS, and animal models to confirm the causal relationship of leptin and leptin bioavailability to AIS. In summary, the findings in this series of studies demonstrated the importance of leptin and leptin bioavailability in skeletal growth, body build, bone quality, and the etiopathogenesis of AIS.