Fructose Upregulates FGF23 Expression In MC3T3 Pre-osteoblasts

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1 Fructose Upregulates FGF23 Expression In MC3T3 Pre-osteoblasts Edek A. Williams, B.S.E., Veronique Douard, Ph.D., Joseph M. Lomuti, B.S., Ronaldo Ferraris, Ph.D., J. C. Fritton, Ph.D.. Rutgers University, Newark, NJ, USA. Disclosures: E.A. Williams: None. V. Douard: None. J.M. Lomuti: None. R. Ferraris: None. J.C. Fritton: None. Introduction: Increased consumption of high fructose has been linked to marked reductions in bone quality in humans and animals [1]. Fructose is directly transported into the blood circulation via transporters in the gut, primarly GLUT5 and metabolized specifically by ketohexokinase (KHK, commonly called fructokinase) [1]. The number of GLUT5 transporters and expression of enzymes for glucose are lower than those used for glucose, an essential sugar consumed for energy. Our data, and that of others, demonstrate that GLUT5 and KHK are expressed in the bone of growing mice [2,3]. Thus, fructose transporters are present in bone, fructose is likely metabolized in bone and fructose consumption disrupts bone formation by osteoblasts [2]. We wish to further understand the cellular mechanisms behind these alterations in bone biology and quality to understand why excessive fructose intake leads to poor bone growth. In this study we tested the hypothesis that bone cells exposed in vitro to fructose up-regulate sugar metabolism and have an anomalous differentiation to the osteoblast phenotype. Our outcome measure was the gene products that determine fructose metabolism in the cell (KHK), and indicate maturation of osteoblasts (osteocalcin and FGF23). Concentrations of fructose were tested from 0 through the physiologic range to supra-physiological. Methods: MC3T3-E1 osteoblasts were cultured for up to 14 days in osteoblast growth media [alpha modified essential media (αmem, containing 5.6 mm/l glucose) with 10% heat inactivated fetal bovine serum, 1% Penicillin/Streptomycin, 0.1% amphotericin b], with 0, 0.25, 0.5 and 1.0 mmol/l fructose to mimic no fructose, moderate fructose, physiological fructose and supra-physiological fructose concentrations in serum. MC3T3 cells were plated at passage 10, expanded and stored at passage 16 prior to seeding onto 12 well plates. After reaching 80% confluence (~4 x 10 5 cells) cells were induced to differentiate to osteoblasts with β-glycerolphosphate and ascorbic acid (media diff ) growth media (day 0). At days 7 and 14, RNA was isolated. mrna extracts were processed for real-time polymerase chain reaction (qpcr) with primers for osteocalcin (OCN), fibroblast growth factor 23 (FGF23) and KHK. Isolated primary cells from long-bone marrow of wild-type C57BL/6 mice aged 3-8 weeks old were treated as described above. After one-week culture, adherent pre-osteoblasts were isolated and induced with media diff. qpcr was performed with bone specific primers for activator of nuclear factor kappa ligand (RANKL), osteoprotegrin (OPG), matrix extracellular phosphoglycoprotein (MEPE), collagen 1, subunit alpha1 (Col1a1), runt related transcription factor 2 (Runx2) and phosphate regulating endonuclease homolog, X-linked (Phex). Sugar transport and metabolism specific primers used in addition to KHK were glucose transporters (GLUT2/5/8/9/12), and triokinase. Elongation factor 1 alpha (EF1α), and 18s ribosomal RNA (18S) were used as housekeeping genes to normalize expression. Results: MC3T3 exposed to fructose for 7 and 14 days at varying concentrations revealed increased expression of FGF23 and OCN (Figures 1-2). Longer exposure resulted in greater increases in gene expression. Surprisingly KHK expression decreased, both with fructose concentration and time (Figure 3). Primary cells demonstrated down-regulated bone-formation markers with fructose exposure (Figure 4). The RANKL/OPG ratio was increased with fructose. Similar to results in MC3T3 data, fructose downregulated the early rate-limiting steps of fructose metabolism, including the expression of glucose transporters 2,5,8,9,12 and KHK, while triokinase, which prepares fructose metabolites to enter the citric cycle, was up-regulated (Figure 5). Discussion: After ingesting 1g/kg BW of fructose, portal vein fructose concentrations approach 0.5 mmol/l but never exceed the concentration of glucose in serum, roughly 10 mmol/l. Thus, the concentrations chosen for this in vitro work represent subphysiological to an upper extreme for serum fructose concentration. In both our MC3T3 and primary cell cultures we observed that the gene products responsible for regulation of mineralized matrix and osteoblast differentiation are diminished in the presence of fructose. Previous work in growing mice challenged with low calcium diet demonstrated increased FGF23 serum levels. Increased FGF23 expression in MC3T3 cells supports what is seen in the serum. In addition to FGF23 expression, KHK expression, an enzyme which converts fructose to fructose-1-phosphate, decreases over time and with fructose concentration. This may indicate that as osteoblasts mature they have reduced capacity for KHK-mediated metabolism. We also observed increased OCN expression. OCN is another bone-specific protein that has a proposed endocrine role in osteoblast energy metabolism. Further characterization of the phenotype and the matrix produced by osteoblasts exposed to fructose is required. Limitations exist in this study. Additional time points should and will be tested in the future. Complete maturation of osteoblasts through collagen matrix production to the expression of proteins that assist in mineralization requires approximately 28 days. Confirmation of osteoblast phenotype may be accomplished with functional assays for alkaline phosphatase (ALP) activity, mineralization (Alizarin Red stain) and collagen (Sirius Red/Fast Green stain). Additionally, GLUT5, RANKL, OPG, MEPE, and Phex expression should be quantified in the MC3t3 cells in parallel with the phenotype confirmatory studies. Severely deficient expression of Phex, a messenger with downstream implications for the degradation of FGF23, could suggest a possible

2 mechanism for increased FGF23 expression in MC3T3 cells. This might support our previous in vivo data [2]. In addition to reduced expression of bone formation markers, the RANKL/OPG ratio was skewed toward that promoting osteoclastogenesis. Further work will be required in a co-culture system to determine the implications of fructose on bone resorption. However, exposure of MC3T3 and primary osteoblasts to fructose has now suggested that FGF23 may be involved in the decreased bone formation seen in our previous in vivo mouse model of mice fed fructose. Significance: High fructose corn syrup has become a staple sweetener in the diets of children. Bone growth is affected by dietary elements. However, few studies have investigated fructose effects on bone growth [1]. Acknowledgments: Funding through NSF: and NIH: AR Technical assistance provided by Mr. Joseph Geissler (NJMS). References: 1. Douard et al. J Physiol 591:401-14, Williams et al. ASBMR, Tsanzi et al. Nutr Rev 66:301-9, 2008.

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6 ORS 2014 Annual Meeting Poster No: 0560

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