Nearly one-third of children in the United States are overweight or obese by their pre-teens. Tall stature and accelerated bone elongation are characteristic features of childhood obesity, which co-occur with conditions such as limb bowing, slipped epiphyses, and fractures. Obese children paradoxically have normal circulating IGF-I, the major growth-stimulating hormone. Here we describe and validate a mouse model of excess dietary fat to examine mechanisms of growth acceleration in obesity. We used in vivo multiphoton imaging and immunostaining to test the hypothesis that high-fat diet increases IGF-I activity and alters growth plate structure before the onset of obesity. We tracked bone and body growth in male and female C57BL/6 mice (N = 114) on high-fat (60% kcal fat) or control (10% kcal fat) diets from weaning (3-weeks) to skeletal maturity (12-weeks). Tibial and tail elongation rates increased after brief (1-2 week) high-fat diet exposure without altering serum IGF-I. Femoral bone density and growth plate size were increased, but growth plates were disorganized in not-yet-obese high-fat diet mice. Multiphoton imaging revealed more IGF-I in the vasculature surrounding growth plates of high-fat diet mice, and increased uptake when vascular levels peaked. High-fat diet growth plates had more activated IGF-I receptors and fewer inhibitory binding proteins, suggesting increased IGF-I bioavailability in growth plates. These results, which parallel pediatric growth patterns, highlight the fundamental role of diet in the earliest stages of developing obesity-related skeletal complications and validate the utility of the model for future studies aimed at determining mechanisms of diet-enhanced bone lengthening.
Rising trends in childhood obesity are related, in part, to increased dietary fat intake. Accelerated bone growth, a hallmark of juvenile obesity but one of its most overlooked complications, can lead to irreversible skeletal damage and chronic adult disability. Paradoxically, obese children typically have low to normal levels of the growth promoting hormone insulin‐like growth factor‐1 (IGF‐1), despite their accelerated growth rate. Obesity is characterized by chronic inflammation driven by cytokines such as interleukin‐6 (IL‐6) which can suppress bone elongation by inhibiting IGF‐1. We previously found that a high‐fat diet increases bone growth and alters serum cytokines in young male and female mice before significant changes in body mass were evident. Our goal here was to determine whether excess body fat could explain the increase in bone elongation rate when overall body mass remains similar. We tested the hypothesis that mice on a high‐fat diet exhibit changes in body composition along with higher levels of inflammatory cytokines before they develop overt obesity. Methods Male 3‐week‐old C57BL/6 mice (N=6/diet) were put on high‐fat (60% kCal fat) or control (10% kCal fat) diet for 2 weeks after weaning. Serum cytokines (Leptin, TNF‐α, IGF‐1, IL‐6, VEGF, IL‐1α, IL‐1β, MCP‐1) were measured by ELISA. Skinfold thickness in the rump region was measured in a separate group of mice using a dial micrometer to estimate body fat. Statistical significance (p<0.05) was determined in SPSS using Mann‐Whitney and t‐tests. Results After only 2 weeks, tibial elongation rate was over 10% higher in mice on a high‐fat diet (t=2.1, p<0.05), but had no difference in body mass (t=0.5, p=0.62). Among the cytokines measured, TNF‐α (z=2.9, p<0.01) and IL‐6 (z=2.7, p<0.01) were both decreased in the high‐fat diet group, while VEGF (z=2.7, p<0.01) was increased. Rump skinfold was nearly 7% greater (t=2.6, p<0.05) in the high‐fat diet group, indicating a higher proportion of body fat. Discussion Our results support the hypothesis that a high‐fat diet alters body composition and inflammatory cytokines before overt signs of obesity. Unexpectedly, the most robust change was a 1.5‐fold decrease in IL‐6, rather than the increase that we had predicted. Since elevated levels of IL‐6 suppress growth, our findings of decreased IL‐6 are consistent with accelerated bone lengthening. IL‐6 also has a role in energy metabolism, and so the changes in body composition that we observed (increased subcutaneous fat) might play a role in altering serum cytokines. Significance These results are relevant for understanding the development of obesity‐related skeletal complications. Reduced inflammatory cytokines such as IL‐6 could serve as a useful biomarker of potential adverse effects, providing the opportunity to implement interventions to mitigate long‐term skeletal damage before it might otherwise be recognized.
Obesity can negatively impact the skeleton. We previously reported that a high‐fat diet causes rapid and dysregulated bone elongation. Our goal was to determine whether a diet intervention could mitigate or even reverse some of these effects on skeletal growth plates. We tested the HYPOTHESIS that rapid and dysregulated growth caused by a high‐fat diet will be mitigated by a low‐fat diet intervention. We expected to see a reduction in growth rate and changes in growth plate morphology that matched controls after switching to a low‐fat diet. METHODS 3‐week old C57BL/6 mice (N=96 mixed sex) were fed control (10% kcal fat) or high‐fat (60% kcal fat) diets for one week. Some high‐fat diet mice were switched to control diet and examined at 5‐, 6‐, and 8‐weeks age (at least N=5/diet/age). Sex by diet interactions were tested at 5‐weeks. Body mass was recorded. Tibial elongation rate was measured from oxytetracycline labeled (7.5 mg/kg IP) slab sections. Growth plate height and columnar orientation (angle of proliferative column relative to long axis) were quantified in ImageJ. Statistical significance (p<0.05) was determined in SPSS by ANOVA with post‐hoc tests. RESULTS There were no sex by diet interactions, so females were analyzed separately to discriminate diet effects. High‐fat and intervention groups were both heavier than controls at 4‐weeks before the intervention (F=7.7, p<.001). Body mass declined sharply after the diet switch and intervention mice were similar to controls at 5‐, 6‐, and 8‐weeks, while high‐fat diet mice remained heavier (p<0.001). Tibial elongation rate was faster in high‐fat diet mice (F=12.2, p<0.001), but slower in the intervention group compared to controls at both 5‐ (p<.001) and 6‐weeks age (p<0.01). Relative to controls, the most significant changes in growth plate height (F=10.7, p<0.001) were enlarged growth plates in high‐fat diet mice at 5‐weeks (p<0.001) and smaller growth plates in intervention mice at 6‐weeks (p=0.003). Chondrocyte columns in controls aligned within 2 degrees of the long axis at 5 weeks, while columns in both high‐fat and intervention mice deviated over 10 degrees (F=17.3, p<0.001). Columns remained disorganized in high‐fat and intervention groups at 6‐ and 8‐weeks (p<0.01). DISCUSSION After switching from high‐ to low‐fat diet, intervention mice exhibited a rapid decrease in body mass, tibial elongation rate, and growth plate height. While we hypothesized that diet intervention would attenuate these to control levels, we surprisingly found that tibial elongation rate in the intervention group was actually slower than that of controls, resembling “inverse catch‐up growth” to achieve a potential target growth rate. However, chondrocyte columns remained disorganized, suggesting a possible lasting consequence of high‐fat diet. SIGNIFICANCE These results demonstrate that the skeleton is exquisitely sensitive to diet in the earliest stages of postnatal bone elongation and are relevant for establishing interventions to prevent childhood obesity and its negative effec...
INTRODUCTION Childhood obesity is driven, in large part, by increased dietary fat intake. Accelerated bone growth, a hallmark of juvenile obesity but one of its most overlooked complications, can lead to irreversible skeletal damage and chronic adult disability. Paradoxically, obese children typically have low to normal circulating levels of the growth promoting hormone insulin‐like growth factor‐1 (IGF‐1), despite their accelerated growth rate. However, IGF‐1 is both an endocrine and paracrine hormone that is essential for endochondral ossification, the process by which bones lengthen in cartilaginous growth plates at the ends of long bones. We previously reported that pre‐obese juvenile mice on a high‐fat diet have increased cell proliferation in growth plates without changes in serum IGF‐1, and before significant changes in body mass were evident. Our goal was to determine whether a high‐fat diet alters local IGF‐1 activity in growth plates by using gene set enrichment analysis on RNA sequencing (RNA‐Seq) data. We tested the HYPOTHESIS that juvenile mice on a high‐fat diet will exhibit an upregulation of IGF‐1 pathways in the growth plate. METHODS Male and female 3‐week old C57BL/6 mice were weaned on to high‐fat diet (60% kCal of fat) or control diets (10% kCal of fat) for 2 weeks and examined at 5‐weeks age when bones are elongating faster in high‐fat diet mice. We developed and validated a manual dissection technique to isolate proximal tibial growth plates from surrounding bone for RNA and protein analyses on cartilage. RNA was isolated from pooled left and right tibial growth plates and sent to the Marshall Genomics Core for next generation sequencing using an Illumina NextSeq 2000 Sequencer. Data were analyzed using fast gene set enrichment analysis (version 1.20.0) along with the reactome pathway database (version 78). Enrichment scores were calculated based on the degree to which genes in a given pathway were up‐ or down‐regulated in the RNA‐Seq data set. These scores are then normalized to account for the different number of genes involved in each pathway set and compared between diets. RESULTS Our data show that pathways regulating IGF‐1 transport and uptake by insulin‐like growth factor binding proteins (IGFBPs) were upregulated in mice on a high‐fat diet with a normalized enrichment score (NES) of 2.17 (Benjamini‐Hochberg adjusted p‐value<0.01). Components of this pathway included, among others, individual IGFBPs (Igfbp4 and Igfbp5) and an IGFBP protease inhibitor (Stc2). DISCUSSION Significant changes in the IGF‐1 pathway closely followed those involved in cell metabolism and muscle contraction, suggesting that regulating IGF‐1 activity could be a main driver of diet‐induced growth acceleration. These data are consistent with our hypothesis that a high‐fat diet increases local IGF‐1 activity in growth plates even though systemic IGF‐1 remains unchanged. SIGNIFICANCE These data are relevant for understanding mechanisms of diet‐induced growth acceleration and provide important groundwork for subsequ...
Insulin-like growth factor-1 (IGF-1) is a critical modulator of cell growth and survival, making it a central part of maintaining essentially every biological system in the body. Knowledge of the intricate mechanisms involved in activating IGF-1 signaling is not only key to understanding basic processes of growth and development, but also for addressing diseases, such as cancer and diabetes. This brief review explores how dysregulation of normal IGF-1 signaling can impact growth by examining its role in postnatal bone elongation. IGF-1 actions are dysregulated in autoimmune diseases, such as juvenile idiopathic arthritis and chronic kidney disease, which results in growth stunting. Conversely, childhood obesity results in growth acceleration, premature growth cessation, and ultimately, diminished bone quality, while systemic IGF-1 levels remain normal. Understanding the role of IGF-1 signaling in normal and dysregulated growth can add to other studies that address how this system regulates chronic diseases.
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