Recent studies have shown that autophagy is essential for proper ␤-cell function and survival. However, it is yet unclear under what pathogenic conditions autophagy is inhibited in ␤-cells. Here, we report that long term exposure to fatty acids and glucose block autophagic flux in ␤-cells, contributing to their toxic effect. INS1 cells expressing GFP-LC3 (an autophagosome marker) were treated with 0.4 mM palmitate, 0.4 mM oleate, and various concentrations of glucose for 22 h. Kinetics of the effect of fatty acids on autophagy showed a biphasic response. During the second phase of autophagy, the size of autophagosomes and the content of autophagosome substrates (GFP-LC3, p62) and endogenous LC3 was increased. During the same phase, fatty acids suppressed autophagic degradation of long lived protein in both INS1 cells and islets. In INS1 cells, palmitate induced a 3-fold decrease in the number and the acidity of Acidic Vesicular Organelles. This decrease was associated with a suppression of hydrolase activity, suppression of endocytosis, and suppression of oxidative phosphorylation. The combination of fatty acids with glucose synergistically suppressed autophagic turnover, concomitantly suppressing insulin secretion. Rapamycin treatment resulted in partial reversal of the inhibition of autophagic flux, the inhibition of insulin secretion, and the increase in cell death. Our results indicate that excess nutrient could impair autophagy in the long term, hence contributing to nutrient-induced ␤-cell dysfunction. This may provide a novel mechanism that connects diet-induced obesity and diabetes.Macroautophagy (hereafter named autophagy) is the main mechanism the cell uses to degrade damaged and redundant organelles. It involves the formation of a double-membrane structure called the phagophore, which evolves into the autophagosome (AP), 2 an organelle that sequesters cytoplasmic material such as mitochondria, peroxisomes, endoplasmic reticulum, protein aggregates, and lipids. Upon acidification (1), the AP fuses with the lysosome to form the autolysosome, which degrades its content (2).The main approach to study autophagy is by tracking APs using LC3 (microtubule-associated protein 1 light chain 3), a cytosolic protein that upon stimulation of autophagy is lipidated and recruited to the AP membrane. LC3 remains bound to the AP until released to the cytosol or degraded by lysosomal enzymes (3).Stimulators of autophagy are known to increase the number of APs. However, the quantification of APs to assess autophagy can be misleading, APs being but one component in the chain constituting autophagic degradation (3). Thus, for example, in the case of various neuronal diseases, the increase in the number of APs was originally falsely interpreted as an increase in autophagic turnover, although it is now known to be the result of a decrease in autophagic turnover downstream to AP formation (4, 5).Type 2 diabetes is a disease in which glucose homeostasis is impaired due to peripheral insulin resistance accompanied by a decrease...