Differential regulation of IGF‐1 pathway and accelerated bone elongation in pre‐obese juvenile mice

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.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.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).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.These data are relevant for understanding mechanisms of diet-induced growth acceleration and provide important groundwork for subsequent efforts to identify specific components of the IGF-1 pathway that could underlie rapid skeletal growth in juvenile obesity.