VITAMIN D MODULATES LIPID STORAGE AND MITOCHONDRIAL FUNCTION IN SKELETAL MUSCLE

Background: Excess adiposity is connected to approximately 1 in 5 deaths in the United States. Many obesity-related diseases are associated with pathological lipotoxicity: a complex set of cellular responses and dysfunctions driven by excessive lipid accumulation in non-adipose tissues. In skeletal muscle, fatty acid accumulation triggers signaling pathways and oxidative stress that lead to insulin resistance and mitochondrial dysfunction. However, vitamin D has been connected to improved insulin sensitivity and reduced lipotoxicity in skeletal muscle. Our lab and others have shown that vitamin D improves lipid distribution in human skeletal muscle. These data point to a vitamin D mediated shift in lipid storage and metabolism that may ameliorate complications of lipotoxicity in skeletal muscle. Methods: C2C12 mouse myoblasts were seeded at high density and differentiated for 3-5 days. Following differentiation, cells were treated with 100 µM palmitate (Palm) or vehicle and 100 nM calcitriol (VitD) or vehicle in a 4-group design for 0, 24, 48, or 72 hours. Gene expression was measured in response to treatment duration via RT-qPCR and lipid storage was measured using oil red O (ORO). Additionally, Seahorse Biosciences XF96 Extracellular Flux Analyzer was used to measure oxygen consumption rate (OCR) in human skeletal muscle cells treated with or without 100 nM VitD for 24 hours. Results: Treatment with both Palm and Palm+VitD produced a time-dependent increase in mean expression of lipid storage genes PLIN2 and PLIN3. The extent of this increase was greater when cells were treated with Palm+VitD (6.6x vs 2.9x), though it did not reach statistical significance. Palm+VitD exhibited a time dependent increase in DGAT (8.9x at 72 h) not seen in the Palm treatment. ORO staining revealed an approximately 5x increase in lipid staining with smaller, more numerous droplets in VitD+Palm treated cells compared to control at all time points, while Palm only matched this increase at 72 h and contained larger lipid droplets. OCR analysis showed that calcitriol treatment in human skeletal muscle increased oxygen consumption at baseline by 57% (p=0.01) and ATP linked OCR by 75% (p=0.03). Conclusions: Our data suggest that Vitamin D mediates beneficial changes in lipid droplet physiology and mitochondrial function. As such, vitamin D may be an inexpensive intervention to prevent cellular stress, lipotoxicity, and insulin resistance. Future work will use siRNA to identify the roles played by PLIN proteins in the observed changes in lipid storage and metabolism.