Tracing and modeling lipid homeostasis to understand keratinocyte biology

Dysregulation of metabolism contributes to the development and progression of many diseases, including cancer. Sphingolipids are bioactive lipids that mediate key cellular functions, such as signaling, apoptosis, and cell proliferation. Previously, we showed the role of serine metabolism in sensitizing tumors through altered sphingolipid biosynthesis to induce metabolic stress, thereby constraining tumor growth. Given the importance of serine, and other nonessential amino acids, in oncogenesis and lipid metabolism, our focus is to explore the tricarboxylic acid (TCA) cycle and sphingolipid metabolic flux in keratinocytes and how these may be dysregulated in cancer. To first evaluate sphingolipid biosynthesis in human keratinocytes, we performed flux measurements in media containing 13 C-serine and 13 C-glycine, which revealed decreased synthesis of ceramides and sphingomyelins in differentiated keratinocytes compared with undifferentiated keratinocytes. Stable isotope tracing with uniformly-labeled 13 C-glucose showed increased flux through the TCA cycle upon differentiation. These data highlight the distinct metabolic changes induced by differentiation and serve as a benchmark for future studies in defining TCA metabolic flux and acyl chain specific alterations to sphingolipid pools in non-melanoma skin cancer models.