Drosophila CerS homeodomain sites differentially alter insulin resistance

Background Ceramides are central sphingolipid intermediates whose abundance and molecular composition are closely linked to insulin resistance. Ceramide synthases (CerSs) generate ceramides and, in several family members, also contain a conserved homeodomain with poorly understood metabolic functions. Drosophila has a single CerS, Schlank, which contains a catalytic Lag1p motif and two predicted nuclear localization sequences, NLS1 and NLS2. We investigated how distinct Schlank homeodomain mutations affect high-sugar diet (HSD)-induced insulin resistance. Results Endogenous KINLS1 and KIE118A knock-in alleles retained DNA binding and transcriptional repression under basal conditions but increased susceptibility to HSD-induced metabolic dysfunction. Both mutants developed enhanced hyperglycemia and impaired insulin-responsive tGPH recruitment, while KIE118A additionally showed increased FoxO nuclear localization and DILP2 retention in insulin-producing cells. By contrast, fat-body-specific switching to the DNA-binding-deficient NLS2 variant or the catalytically inactive H215D variant attenuated selected HSD-induced phenotypes, including FoxO nuclear accumulation and DILP2 retention, without fully correcting hyperglycemia or reduced body weight. Lipidomic analysis revealed extensive diet-dependent sphingolipid remodeling. In particular, NLS2 and H215D markedly reduced the fat-body ceramide pool, including abundant d14:1/20:0- and d14:1/22:0-containing Cer, CerPE, and HexCer species. Conclusions The Schlank homeodomain regulates insulin homeostasis in a site- and tissue-dependent manner. Distinct homeodomain mutations can either increase susceptibility to or partially attenuate HSD-induced insulin resistance, potentially involving insulin signaling, inter-organ communication, and sphingolipid composition.