Aims/hypothesis: Chronic exposure of pancreatic beta cells to high glucose and fatty acids has been proposed to induce glucolipotoxicity. However, contradictory results suggest adaptations of the beta cells, which might be instrumental for partial preservation of the secretory response. In this context, we delineated the expression pattern of genes related to lipid pathways along with fat storage/mobilisation during glucose-stimulated insulin secretion. Methods: Insulin-secreting cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mmol/l) without or with BSA-complexed 0.4 mmol/l palmitate and oleate. Then, transcriptomic analyses of lipid pathways were performed in human islets by RNA-Seq and in INS-1E cells and rat islets by quantitative RT-PCR. Storage of fat was assessed in INS-1E cells by electron microscopy and Bodipy staining, which was also used for measuring lipid mobilisation rate. The secretory response was monitored during acute 15 mmol/l glucose stimulation using online luminescence assay for INS-1E cells and by radioimmunoassay for rat islets. Results: In human islets, chronic exposure to palmitate and oleate modified expression of a panel of genes involved in lipid handling. Culture at 25 mmol/l glucose upregulated genes encoding for enzymes of the glycerolipid/NEFA cycle and downregulated receptors implicated in fatty acid signalling. Similar results were obtained in INS-1E cells, indicating enhanced capacity of the glycerolipid/NEFA cycle under glucotoxic conditions. Exposure to unsaturated C18:1 fatty acid favoured intracellular lipid accumulation in a glucose-dependent way, an effect also observed with saturated C16:0 fatty acid when combined with the panlipase inhibitor Orlistat. After the glucolipotoxic culture, intracellular fat mobilisation was required for acute glucose-stimulated secretion, particularly in oleate-treated cells under glucotoxic culture conditions. The lipid mobilisation rate was governed chiefly by the levels of stored fat as a direct consequence of the culture conditions rather than energetic demands, except in palmitate-loaded cells. Conclusions/interpretation: Glucolipotoxic conditions promote the capacity of the glycerolipid/NEFA cycle thereby preserving part of the secretory response. The cycle of fat storage/mobilisation emerges as a mechanism helping the beta cell to cope with glucotoxic conditions.