Hepatocyte Serine Palmitoyltransferase 3 is essential for mitochondrial ceramide homeostasis and electron transport chain function.

Sphingolipids constitute essential membrane lipids that also function as signaling molecules mediating fundamental cellular processes. Disruption of constitutive sphingolipid metabolism contributes to a range of metabolic diseases. Sphingolipid biosynthesis is initiated by serine palmitoyltransferase (SPT), a heteromeric enzyme composed of Sptlc1 and either Sptlc2 or the less-characterized subunit Sptlc3. While the canonical Sptlc1/Sptlc2 complex generates most sphingolipids, Sptlc1/Sptlc3 produces non-canonical sphingolipids that remain poorly understood. Sptlc3 expression increases in liver disease in both mouse models and humans, yet its biological role remains unclear. To address this, we generated a liver-specific SPTLC3 knockout (SPT3-hKO) mouse model. Loss of SPTLC3 resulted in severe mitochondrial dysfunction in hepatocytes, characterized by reduced oxygen consumption, decreased ATP production, and elevated NADH/NAD+ ratios with a concomitant increase in glycolytic activity. Mechanistically, we found a defect in the electron transport chain (ETC) which stemmed not from intrinsic impairment of the ETC complexes but rather from hindered electron flow between complex I and complex III likely due to reduced availability of Coenzyme Q. While atypical ceramides constituted (∼4% of total ceramides) when measured in liver homogenates, there measurements in mitochondria-enriched fractions revealed enrichment in this cell compartment. Together, our findings identify SPTLC3 as a critical modulator of mitochondrial ceramide composition and ETC function, revealing a specialized role for non-canonical sphingolipids in hepatocyte energy metabolism.