Lipolytic breakdown of endogenous lipid pools in pancreatic β-cells contributes to glucose stimulated insulin secretion (GSIS). Recently, it has been shown in other cell types, that endogenous lipid can be metabolised by autophagy, where macromolecules are delivered to the lysosome for degradation. This lipophagy is catalysed by lysosomal acid lipase (LAL). Our current aim was to investigate LAL in β-cells, and its role in lipophagy and GSIS.
Measuring insulin secretion by RIA, GSIS was upregulated 1.8 fold in MIN6 cells pretreated for 24hr with Lalistat (LAL inhibitor) (n=4; P<0.05). Isolated mouse islets also showed increased insulin secretion following Lalistat pretreatment. Interestingly, this increase was inhibited by acute addition of Orlistat (pan-lipase inhibitor), indicating that acute mobilisation of stored lipid is required for this enhancement. As expected, Lalistat treatment of MIN6 cells increased neutral lipid pools, measured by lipid GC-MS (n=4): cholesterol ester 6.4 fold (P<0.001), triacylglycerol 1.7 fold (P<0.05) and diacylglycerol 2 fold (P<0.05). Confocal microscopy showed increased neutral lipid staining in Lalistat treated MIN6 cells, and co-staining with LAL indicated that the increased lipid was not lysosomally located. Lalistat treatment also decreased LC3-II (autophagosome marker) accumulation following chloroquine treatment, indicating potential cross-talk between LAL activity and autophagy, and had no effect on overall lysosomal function. Therefore, we inhibited autophagy using atg7 siRNA in MIN6 cells, which caused a 1.7 fold increase in GSIS (n=4; P<0.05). In mouse islets treated with 5mmol/L 3-methyladenine for 24hr to inhibit autophagy, GSIS increased 2.4 fold (n=6; p<0.0001), this effect was partially blunted by Orlistat (n=6; p<0.05), highlighting a specific role for lipophagy in regulating insulin release from mouse islets.
Our data suggests a novel pathway of lysosomal lipid degradation, utilising LAL and lipophagy, is a constitutive negative regulator of GSIS. This process is most likely mediated by depletion of substrate for the neutral lipases that are activated by glucose.