Leptin signaling and the intervertebral disc: Sex dependent effects of leptin receptor deficiency and Western diet on the spine in a type 2 diabetes mouse model

Abstract

Type 2 diabetes and obesity are associated with back pain in juveniles and adults and are implicated in intervertebral disc (IVD) degeneration. Hypercaloric Western diets are associated with both obesity and type 2 diabetes. The objective of this study was to determine if obesity and type 2 diabetes result in spinal pathology in a sex-specific manner using in vivo diabetic and dietary mouse models. Leptin is an appetite-regulating hormone, and its deficiency leads to polyphagia, resulting in obesity and diabetes. Leptin is also associated with IVD degeneration, and increased expression of its receptor was identified in degenerated IVDs. We used young, leptin receptor deficient (Db/Db) mice to mimic the effect of diet and diabetes on adolescents. Db/Db and Control mice were fed either Western or Control diets, and were sacrificed at 3 months of age. Db/Db mice were obese, while only female mice developed diabetes. Female Db/Db mice displayed altered IVD morphology, with increased intradiscal notochordal band area, suggesting delayed IVD cell proliferation and differentiation, rather than IVD degeneration. Motion segments from Db/Db mice exhibited increased failure risk with decreased torsional failure strength. Db/Db mice also had inferior bone quality, which was most prominent in females. We conclude that obesity and diabetes due to impaired leptin signaling contribute to pathological changes in vertebrae, as well as an immature IVD phenotype, particularly of females, suggesting a sex-dependent role of leptin in the spine.

Fig 1. Study design.

A) Leptin receptor deficient mice and their heterozygous controls were fed either a Western Diet or Control Diet diet. Mice were sacrificed at 12 weeks. B) Dietary information.

Fig 2. Leptin receptor deficiency and Western diet both increased fasting body weight in mice, for both male and female groups.

p < 0.5. wk = weeks.

Fig 3. Leptin receptor deficiency increased HbA1c in mice for both male and female groups.

A) HbA1c was elevated to diabetic levels in female mice. B) Blood glucose did not reach diabetic levels. p < 0.5. wk = weeks.

Fig 4. Trabecular bone increased with leptin receptor deficiency.

A) 3D μCT images of trabecular bone of (top) female and (bottom) male mice. B) BV/TV C) Tb.Sp, and D) Tb.N demonstrate inferior Tb microstructure with Db/Db genotype. Only female Db/Db mice had E) increased Tb. BMD and F) decreased Tb.Th. p < 0.05. BV/TV = bone volume fraction; Tb.Sp = trabecular spacing; Tb.N–trabecular number; Tb. BMD = trabecular bone mineral density; Tb.Th = trabecular thickness.

Fig 5. Leptin receptor deficiency caused decrease in cortical bone mainly in female mice.

A) 3D μCT images of cortical bone for (top) female and (bottom) male mice. Two-way ANOVA revealed that, in female mice, leptin receptor deficiency decreased B) Ct.Ar/Tt.Ar C) Ct.Ar and D) Ct.Th. While increasing G) Ct.BS/BV. Post Hoc testing indicated that this effect was only significant in female mice on WD. No changes were observed for E) Tt.Ar or F) Ct.TMD. In male 2-way ANOVA analysis indicated a diet effect in Ct.Ar and a genotype effect in Ct.Th and Ct. BS/BV. Post Hoc testing indicated that increased Ct.BS/BV was only significant in male mice on WD p < 0.05. Ct.Ar/Tt.Ar = cortical area fraction; Ct.Ar = cortical area; Ct.Th = cortical thickness; Tt.Ar = total area; Ct. TMD cortical tissue mineral density.

Fig 6. Notochordal band size increased in female leptin receptor deficient mice.

A) Representative Picrosirius red/Alcian blue images demonstrate an increased notochordal band in (top) female Db/Db mice. B) Db/Db genotype did not cause IVD degeneration. In female Db/Db mice, C) notochordal cell size was increased and D) cells per area was decreased. No changes were observed in male IVDs. Black boxes mark region of interest (ROI).

Fig 7. Leptin receptor deficiency caused decrease vertebral length and disc height, but no change in disc height index.

A) Schematic of IVD height and vertebrae length measurement. B) μCT midsagittal sections of vertebral bone for (top) female and (bottom) male. p < 0.05. C) vertebral length D) IVD height E) DHI.

Fig 8. Torsional strength decreased with Db/Db genotype.

Schematic curves from A) axial-compression and torsion testing, B) Creep, and C) torsion to failure curves after biomechanical testing analysis. D) torsional stiffness E) Failure strength F) angle to failure. p<0.05.