146 - New Insights into the Role of the IGF-1 Pathway in Diet-induced Bone Elongation

Poster Board Number: 146
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Abstract Body : Introduction: Excess dietary fat intake is a major driver of childhood obesity and can have lasting effects on the growing skeleton. We have previously shown that a high-fat diet increases bone elongation rate in juvenile mice before the onset of obesity. We have also shown that this growth acceleration can be reversed by switching mice back to a low-fat diet. The cause of such rapid, diet-induced growth modulation is still unclear. Using next generation RNA sequencing, we found 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. We used immunostaining to demonstrate that IGFBP-4, the major IGFBP in bone and cartilage and an IGF-1 inhibitor, is reduced in high-fat diet growth plates. Here we examined a component of IGFBP-4 regulation, pregnancy-associated plasma protein A (PAPP-A) in the proximal tibia. The protease PAPP-A is required to cleave IGFBP-4 to free IGF-1 for biological activity. We tested the hypothesis that high-fat diet feeding increases PAPP-A expression in rapidly elongating juvenile mouse bones.

Methods: Female 3-week old C57BL/6 mice (N=15) were fed control (10% kcal fat) or high-fat (60% kcal fat) diets after weaning. After one week, some high-fat diet mice (N=5) were switched to a control diet (intervention) and all groups were examined at a 6-week age endpoint. Proximal tibiae were collected and bisected. Tibial elongation rate was quantified from oxytetracycline labeled (7.5 mg/kg IP) unfixed slab sections imaged (2.5x) under UV excitation. The other tibial half was fixed, decalcified, sectioned, and stained with a commercially available fluorescently-conjugated primary antibody against PAPP-A (Alexa 647, 1:100, G-Biosciences ITN2299). Fluorescence images (20x) were captured in the epiphyseal marrow, where prominent PAPP-A staining was observed and could be reliably measured. PAPP-A expression was quantified as mean fluorescence intensity in 125 x 100 µm regions of interest. Data were collected from N=4 individuals per group due to tissue quality. Statistical testing (p< 0.05) was performed using ANOVA with Tukey’s post-hoc testing, Kruskal-Wallis, and Spearman’s one-tailed tests in SPSS.

Results: There were significant diet-related differences in PAPP-A staining using both parametric (ANOVA F = 3.6, p< 0.05) and non-parametric approaches (Kruskal-Wallis H = 5.7, p< 0.05), given the small sample size. PAPP-A fluorescence intensities in the high-fat diet mice, whose bones were elongating the fastest, were nearly three-fold greater than those of the intervention mice, whose bones were elongating the slowest (p< 0.05). PAPP-A fluorescence was positively correlated with tibial elongation rate (Spearman’s rho = 0.52, p< 0.05). Surprisingly, the most robust PAPP-A staining was detected in the epiphyseal and metaphyseal marrow, suggesting that IGF-1 bioavailability in the growth plate could be regulated by IGFBP-4 proteolysis in adjacent tissues.

Significance: These data provide unique insight into specific components and locations of the IGF-1 pathway that are sensitive to diet and could underlie rapid skeletal growth in juvenile obesity.