- Short report
- Open Access
Leptin-deficient mice have altered three-dimensional growth plate histomorphometry
© The Author(s) 2019
- Received: 5 October 2018
- Accepted: 17 January 2019
- Published: 24 January 2019
Leptin is an adipokine that regulates energy homeostasis and is also needed for normal bone growth and maintenance. Mutation in the lep gene, which characterizes the ob/ob mouse model, results in the development of obesity and type 2 diabetes mellitus, as well as reduced limb bone length and increased fracture risk. However, the relationship between limb bone length and growth plate cartilage structure in obese diabetic adolescents is incompletely understood. Here, we tested the hypothesis that leptin deficiency affects the microstructure of growth plate cartilage in juvenile ob/ob mice.
Tibial growth plate cartilage structure was compared between lean and obese, leptin-deficient (ob/ob) female mice aged 10 weeks. We used confocal laser scanning microscopy to assess 3D histological differences in Z stacks of growth plate cartilage at 0.2 µm intervals, 80–100 µm in depth. Histomorphometric comparisons were made between juvenile lean and ob/ob mice.
We found obese mice have significantly reduced tibial length and growth plate height in comparison with lean mice (P < 0.05). Obese mice also have fewer chondrocyte columns in growth plate cartilage with reduced chondrocyte cell volumes relative to lean mice (P < 0.05).
These data help explicate the relationship between growth plate cartilage structure and bone health in obese diabetic juvenile mice. Our findings suggest obesity and diabetes may adversely affect growth plate cartilage structure.
- Ob/ob mice
Obesity and type 2 diabetes mellitus (T2DM) are on the rise worldwide in children and adolescents [1, 2]. T2DM now accounts for 45% of all new onset diabetes cases in juveniles, up from 3% just two decades ago . This is a serious health concern as T2DM adversely affects growth and development, resulting in long-term, disabling complications . Once such complication is impaired bone formation and mineralization, which is associated with decelerated bone growth, increased risk of bone fracture, and delayed fracture repair . Impairment of bone formation is caused, at least in part, by leptin dysregulation [3–5]. Leptin is an adipokine secreted primarily by adipocytes to regulate energy homeostasis, but it also plays a role in regulating bone metabolism [6, 7]. Ob/ob mice, which are leptin deficient due to a lep gene mutation, are hyperphagic and exhibit metabolic signatures consistent with the T2DM phenotype . Ob/ob mice also have significantly reduce bone mineral density and shorter limb bones than age-matched wild type mice [9–11].
Longitudinal growth of long bones occurs via endochondral ossification. During this process, growth plate cartilage expands and is replaced with bone tissue. Proliferation, differentiation, and metabolic activity of chondrocytes in the growth plate are inhibited in the obese, T2DM condition . Leptin-deficient mice have growth plates that are reduced in height, likely due to the downregulation of genes regulating ossification [10, 13], although the specific effects of leptin-deficiency on three-dimensional growth plate structure remain unclear. In this study, we used three dimensional histomorphometric analysis to compare long bone growth plate microstructure in lean and leptin-deficient ob/ob mice. The goal was to elucidate the effects of leptin deficiency on growth plate morphology and to improve our understanding of the relationship between growth plate structure and long bone growth in ob/ob mice.
Female obese ob/ob mice (n = 5) and lean +/+ mice (n = 5) of the strain C57Bl/6-Lepob aged 4–5 weeks were purchased for the study (Jackson Laboratory; Bar Harbor, ME, USA). Ob/ob mice in this age range demonstrate obesity and hyperglycemia, as well as reduced thickness of the growth plate [10, 11]. All animals were housed in a facility with a 12 h light/dark cycle at a temperature of 22 °C. Mice were given ad libitum access to standard rodent chow and drinking water, and were treated in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Use of animals was approved by the Institutional Animal Care and Use Committee at Midwestern University.
Statistical comparisons of histomorphometric data were made using SPSS Statistics 25 (IBM, USA). Unpaired two-sampled t tests were performed with statistical significance set at P < 0.05. Kolmogorov–Smirnov and Levene’s tests were used to ensure assumptions of normality and equality of variances were not violated. Data are present as mean ± standard error.
Comparisons of body mass and tibial dimensions in lean (+/+) and obese (ob/ob) mice
Body mass at 5 weeks (g)
21.7 ± 0.64
22.1 ± 0.85
Body mass at 10 weeks (g)
23.4 ± 0.34
24.8 ± 0.95
Tibia length (mm)
17.9 ± 0.11
17.2 ± 0.61
Growth plate height (mm)
1.58 ± 0.12
0.84 ± 0.22
Chondrocyte column count
53.9 ± 8.2
43.6 ± 2.7
Ob/ob mice are leptin deficient and exhibit metabolic profiles similar to obese T2DM patients. However, while ob/ob mice are unable to produce leptin, obese T2DM patients are often hyperleptinemic but demonstrate leptin resistance with similar metabolic consequences as leptin deficiency in ob/ob mice [15–18]. One of these consequences is reduced long bone mass. Like obese T2DM humans, ob/ob mice have a greater risk of bone fracture, along with significantly shorter long bones [9, 11, 19, 20]. Here, we report on growth plate structure in the tibias of ob/ob mice to assess structural differences that may correlate with reduced bone length.
It should be noted that T2DM is known to affect the sexes differently. For example, females with T2DM are more likely to develop myocardial dysfunction and die from heart failure than males . Females also respond more slowly and less robustly to the administration of exogenous leptin and insulin . While we previously found reduced growth plate cartilage thickness and area in male and female mice [10, 13], it is unclear if three-dimensional microstructure differences between the sexes exist. Based on our previous findings, we hypothesize that growth plates of male ob/ob mice will exhibit similar differences in growth plate microstructure as lean controls, but further study is needed to evaluate this hypothesis. Additionally, serum leptin declines more quickly in females than males as they age, independent of body mass index and age-related endocrine changes . There may be sex-related differences in the long-term implications of leptin resistance in children and adolescents with T2DM. Again, further study is needed to validate this prediction.
Our data are in agreement with the mounting evidence that leptin deficiency has a significant impact on the length of long bones in ob/ob mice by affecting growth plate cartilage [9–11, 21]. Our study supports the hypothesis that growth plate morphology in long bones is altered in obese, leptin-deficient mice. Our findings indicate growth plate cartilage exhibits atypical morphology with reduced cell volume and numbers of proliferating cell columns. Given the rise of obesity and T2DM in juveniles, further study is needed to elucidate the clinical implications of leptin dysregulation on long-term bone and joint health.
LA, TLB and JHP contributed to planning the experiments. JH and MC conducted the experimental analyses. JH and JHP drafted the manuscript. All other authors edited and revised the manuscript. All authors read and approved the final manuscript.
Acknowledgements and funding
The authors wish to acknowledge Midwestern University, Diabetes Action Research and Education Foundation, and Soy Health Research Program for funding the study.
The authors declare that they have no competing interests.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The protocol for this study was approved by the Midwestern University Institution Animal Care and Use Committee.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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