Ceramides are a dynamic group of bioactive lipids and major constituents of the epidermal permeability barrier. Alterations in their levels have been reported in skin conditions as well as in metabolic disorders with manifestations in the skin, such as diabetes. Polyunsaturated fatty acids (PUFAs) have been shown to affect ceramide concentration, the underlying mechanisms, however, require further investigation. Aims: This project aims to explore mechanisms involved in the metabolism of cutaneous lipids in normal skin cells treated with PUFAs, and to examine differences in cutaneous and systemic bioactive lipid levels in a streptozotocin (STZ) -diabetic rat model of peripheral neuropathy when compared with control. Methods: Proliferating and differentiating primary normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (HDF) were treated with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and linoleic acid (LA) (10 ÃÂµM). Liquid chromatography coupled to mass spectrometry (UPLC-MS/MS) was used to measure ceramides (CERs), sterols, glycerolipids, free fatty acids (FFAs), glycerophospholipids, sphingomyelins (SM), endocannabinoids, N-acyl ethanolamines and monoacylglycerols. Gene expression of ceramide synthases 1-6 (CERS1-6) and dihydroceramide desaturases 1-2 (DES1-2), was achieved by RT-qPCR. Next generation sequencing was performed to determine the expression of lipid metabolism genes in differentiated and proliferating NHEK. Results: Ceramide concentration was increased upon NHEK differentiation. CER signalling pathways and metabolism genes including serine palmitoyltransferase (SPT) and glucosylceramide, in addition to FFA and triacylglycerol (TG) metabolism genes were highly expressed in differentiated NHEK. Supplementation with DHA (10ÃÂµM) increased ceramide CER[NS] concentration in proliferating and differentiated NHEK, and upregulated CERS3 and DES2 gene expression in proliferating NHEK. DHA reduced SM and TG levels, while EPA reduced FFA levels in proliferating NHEK at 72 hours post-supplementation. Neither n-3 PUFA tested altered gene expression or membrane lipid levels in differentiated NHEK. LA increased some CERs, FFA and lysophosphatidylcholine (LPC) species in differentiated NHEK, but did not affect CER gene expression. Both EPA and DHA increased some CER species in HDF, and downregulated CERS5 and CERS6 mRNA expression. DHA also affected the levels of SM, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species. LA only had an effect on membrane lipids where it reduced cholesterol ester (CE), TG, PC and LPC levels in HDF. In the STZ-diabetic rat model, CER[NH] ceramides were significantly reduced in diabetic skin while short-chain ceramides were increased in plasma, 16 and 12 weeks post STZ, respectively. Significant decreases in palmitoleoyl ethanolamine (POEA) and vaccenoyl ethanolamine (VEA) levels in plasma, and pentadecanoyl ethanolamine (PDEA), oleoyl ethanolamine (OEA) and VEA in skin, were also observed. Conclusion: This study demonstrates the differential effect of n-3 and n-6 PUFA supplementation on CER metabolism in human epidermal and dermal cells. This suggests that DHA may have a potential therapeutic benefit in the epidermis as it increased CER levels in cells representing the basal and granular layers, whereas LA may be essential for improving skin barrier function as it mainly enhanced CER concentration in differentiating cells. Based on these findings, future studies could focus on examining the effect of DHA and LA on the glucosylceramide recycling pathway in differentiated keratinocytes. In addition, investigating the effect of DHA on the SM degradation pathway in the dermis, could help to better understand the mechanisms by which DHA could alter dermal CER levels. The effect of LA on HDF lipids suggests that LA stimulates fibroblast growth, which is in agreement with previous reports on the role of n-6 PUFA in wound healing. Finally, the barrier critical CER[NH] ceramides were reduced in diabetic rat skin with disease progression; Future studies could investigate their potential role in diabetic dermopathies.