Normal leptin expression, lower adipogenic ability, decreased leptin receptor and hyposensitivity to Leptin in Adolescent Idiopathic Scoliosis

Abstract

Leptin has been suggested to play a role in the etiology of Adolescent Idiopathic Scoliosis (AIS), however, the leptin levels in AIS girls are still a discrepancy, and no in vitro study of leptin in AIS is reported. We took a series of case-control studies, trying to understand whether Leptin gene polymorphisms are involved in the etiology of the AIS or the change in leptin level is a secondary event, to assess the level of leptin receptor, and to evaluate the differences of response to leptin between AIS cases and controls. We screened all exons of Leptin gene in 45 cases and 45 controls and selected six tag SNPs to cover all the observed variations. Association analysis in 446 AIS patients and 550 healthy controls showed no association between the polymorphisms of Leptin gene and susceptibility/severity to AIS. Moreover, adipogenesis assay of bone mesenchymal stem cells (MSCs) suggested that the adipogenic ability of MSCs from AIS girls was lower than controls. After adjusting the differentiation rate, expressions of leptin and leptin receptor were similar between two groups. Meanwhile, osteogenesis assay of MSC showed the leptin level was similar after adjusting the differentiation rate, but the leptin receptor level was decreased in induced AIS osteoblasts. Immunocytochemistry and western blot analysis showed less leptin receptors expressed in AIS group. Furthermore, factorial designed studies with adipogenesis and osteogenesis revealed that the MSCs from patients have no response to leptin treatment. Our results suggested that Leptin gene variations are not associated with AIS and low serum leptin probably is a secondary outcome which may be related to the low capability of adipogenesis in AIS. The decreased leptin receptor levels may lead to the hyposensitivity to leptin. These findings implied that abnormal peripheral leptin signaling plays an important role in the pathological mechanism of AIS.

Figure 1. Linkage disequilibrium (LD) pattern and Haplotypes of the Leptin gene from the 45 healthy subjects.

Two LD blocks were identified in the sequenced genomic region of the gene (calculated with the Solid spine of LD algorithm with the Minor Allele Frequency≥1%). Arrows indicate the positions of the 6 tag SNPs selected for the association study.

Figure 2. Relative levels of the genes in induced adipocytes from AIS group versus control group.

The expression of target genes was measured by real-time quantitative RT-PCR and normalized to GAPDH expression. Relative expression levels were calculated by using the 2^−ΔΔCt method. The total group included the whole data set whereas the female and male groups included the data from each gender only. *P<0.05 was considered statistically significant. **P<0.01. All P values were calculated with age adjustment.

Figure 3. Relative levels of the genes in induced osteoblasts from AIS group versus control group.

The expression of target genes was measured by real-time quantitative RT-PCR and normalized to GAPDH expression. Relative expression levels were calculated by using the 2^−ΔΔCt method. *P<0.05 was considered statistically significant. **P<0.01. All P values were calculated with age adjustment.

Figure 4. The expression of leptin receptor in induced adipocytes was determined by immunocytochemistry.

The AIS MSCs (a) was from a 14-year-old boy (Cobb angle = 80°) and the normal control (b) was from a 19-year-old boy. Note that the leptin receptors were abundant in undifferentiated MSCs from control sample, but were rare in those from AIS sample. Scale bar = 200 µm.

Figure 5. Western blot was performed to detect Leptin receptors in adipogenic and osteogenic MSCs.

Two variants of Leptin receptors were present at ∼100 kD and ∼125 kD. The normal control sample (Ctrl) was from a 20-year-old girl and the AIS sample (AIS) was from a 15-year-old girl (Cobb angel = 68°). The level of leptin receptor was obviously lower in AIS sample.

Figure 6. The effect of leptin on adipogenesis of MSCs from a normal control (18y, male) and an AIS patient (15y, male, Cobb angle = 54°).

Cells from the normal control were induced along the adipogenic differentiation with human recombined leptin protein at concentration of 0 µg/mL (a), 0.015 µg/mL (b) and 0.6 µg/mL (c). MSCs from the AIS patient were also treated under the same condition that containing leptin of 0 µg/mL (d), 0.015 µg/mL (e) and 0.6 µg/mL (f). At day 12, the cells were subjected to Oil Red O staining. Scale bar = 500 µm.

Figure 7. Gene expression levels in response to different doses of leptin.

The expression levels in control group without leptin treatment were used as calibrators. Relative expression levels were calculated by using the 2^−ΔΔCt method.

Figure 8. The effect of leptin on osteogenesis of MSCs from a normal control (22y, male) and an AIS patient (18y, male, Cobb angle = 88°).

Cells from the normal control were induced along the osteogenic differentiation with human recombine leptin protein at concentration of 0 µg/mL (a), 0.015 µg/mL (b) and 0.6 µg/mL (c). MSCs from the AIS patient were also treated under the same condition that contained leptin of 0 µg/mL (d), 0.015 µg/mL (e) and 0.6 µg/mL (f). At day 21, the cells were subjected to Alizarin Red S staining. Scale bar = 500 µm.

Figure 9. Gene expression levels in response to different doses of leptin.

The expression levels in control group without leptin treatment were used as calibrators. Relative expression levels were calculated by using the 2^−ΔΔCt method.