Impact of Calcitriol on Fibroblast Growth Factor-23 (Fgf-23) Signaling in Diabetic Dysregulation in Cardiac Fatty Acid and Glucose Metabolism

Background: Cardiovascular disease is the leading cause of diabetes mellitus (DM) related morbidity and mortality. Vitamin D deficiency was observed to induce cardiac remodeling and has been linked with DM. Our previous investigation demonstrated that DM rats had impaired 25(OH)D level resulting in myocardial dysfunction. Calcitriol was found to modulate protein expressions of regulatory enzymes involved in fatty acid metabolism, pro-inflammatory cytokines, PPARs isoforms in DM hearts. However, the direct effect of calcitriol in myocardial energy metabolism, molecular mechanisms resulting in the changes of these protein expressions and signaling pathway underlying the activity of calcitriol in the myocardial inflammatory cytokines, fatty acid metabolism and PPARs expressions has not been fully elucidated. Fibroblast growth factor 23 (FGF-23) is a hormone released by the osteocytes that plays a role in the regulation of phosphate and vitamin D homeostasis through direct actions in the kidney and parathyroid glands. Elevated FGF-23 was found to be correlated with increased fat mass, bigger waist circumference, and adverse abnormalities in lipid profiles in a cohort investigation. However, whether FGF-23 exerts metabolic effect beyond mineral metabolism remained unknown. Moreover, whether FGF-23 is simply marker of cardiac disease risk or direct mediator of cardiac pathology and cardiac performance needs further evaluation. Therefore, the purpose of this study is to investigate the role of calcitriol on the FGF-23 signaling in DM cardiac metabolism. In the first year experiment, we will study the effect of calcitriol on the FGF-23 signaling in the cardiomyocyte energy metabolism. In the second year experiment, we will evaluate the in vivo effect of calcitriol on myocardial fatty acid metabolism and the role FGF-23 signaling in type 2 DM rodent hearts. Methods: First year: Western blot for PPARs isoform, pro-inflammatory cytokines (TNF-α,TGF-β, IL-6), oxidative stress (NOX4, p22phox), fatty acid substrates (AMPK-2, pAMPK-2, pACC, CPT1, PGC1-, CD 36, DGAT 2, DGAT1), down-steam signaling pathways involving calcineurin NFAT, P38 MAPK, ERK1/2, CaMK II kinase, and real-time PCR for FGF receptor (FGFR)1,  Klotho, PPAR isoforms, TNF-, and IL-6 will be studied in the control, hyperglycemic (incubated with D-glucose, 33 mM) and hyperlipidemic (incubated with sodium palmitate, 0.25 mM) HL-1 cells or H9c2 cells treated with FGF-23 (25 ng/ml), FGF-23 with calcitriol (10 nM), and FGFR1 inhibitor (PD-166866, 50 nM) with or without calcitriol (10 nM) for 48 hours. Second year: Non-invasive micro PET scan will be used to evaluate the in vivo efficacy of calcitriol on myocardial energy metabolism in control, type 2 DM rat (high-fat diet combined with low dose streptozotocin), type 2 DM treated with FGF-23 (4 μg/kg, daily, intraperitoneally for 5 days), type 2 DM treated with FGF-23 and calcitriol (cholecalciferol 125 μg/kg, intraperitoneally daily for 5 days). Histopathological examination, immunochemistry, western blot and real time PCR for cardiac PPAR isoforms, myocardial fatty acid substrates (AMPK-2, pAMPK-2, pACC, CPT1, PGC1-, CD 36, DGAT 2, DGAT1), pro-inflammatory cytokines (TNF-α,TGF-β, IL-6), oxidative stress (NOX4, p22phox), RAGE, and GLUT4, GLUT 2, and downstream signaling pathways primarily involving calcineurin NFAT, P38 MAPK, ERK1/2, CaMK II kinase will be examined in isolated tissue preparations. Expected Results: Calcitriol modulate FGF-23 signaling through calcineurin-NFAT signaling in coordination with MAPK signaling pathway