Mo1783 Class III PI3K (HVPS34) Is Involved in AMPK-Regulated Neurotensin Secretion Independent of mTORC1 Signaling

Abstract

Class III PI3K, also known as hVps34, along with its associated regulatory subunit, hVps15 kinase, are critical components of nutrient sensing, and act as upstream factors required for activation of S6K1, a downstream effector of mTOR complex 1 (mTORC1). The hVps34/ hVps15 complex regulates a variety of cellular functions such as intracellular vesicle trafficking. In addition, AMPK regulates the hVps34-containing complex in response to changes in energy state. Neurotensin (NT), a gut hormone produced and stored in N cells of the distal small bowel, has multiple physiologic effects, including facilitation of fatty acid translocation from the intestinal lumen, coordination of gut motility and stimulation of pancreaticobiliary secretion. Since we have previously found that AMPK and mTORC1 positively and negatively regulate NT release, respectively, the purpose of the present study was to investigate whether hVps34 and hVps15 are required in AMPK or mTORC1-mediated secretion. METHODS. i) The human endocrine cell line, BON, was used for all experiments. BON cells synthesize and secrete NT peptide and process the NT peptide in a manner analogous to that of N cells in the small bowel, thus serving as a useful model for enteroendocrine cell secretion. AMPK activation was induced by Aicar or 2-DG, and NT secretion measured using an NT EIA kit. ii) To determine the involvement of hVps34 and hVps15 in AMPK-mediated effects on NT secretion, endogenous expression of hVps34 and hVps15 was inhibited by siRNA-mediated knockdown. Stable BON cell lines overexpressing either the control vector or hVps34/hVps15 (hVps34 and hVps15 are expressed in one vector but regulated by individual promoters) were established. iii) The physical association of hVps34 with AMPK was evaluated by co-immunoprecipitation (co-IP). RESULTS. NT secretion (induced by Aicar or 2-DG) was attenuated in BON cells transfected with siRNAs targeting hVps34 and hVps15 compared to non-targeting control (NTC) siRNA. Overexpression of hVps34/hVps15 increased both basal and Aicarand 2-DG-stimulated NT secretion. Moreover, we detected the physical association of hVps34 with AMPK by co-IP. hVps34 was detected in both AMPKα1and α2-precipitated complexes with increased hVps34 bound in the AMPKα2 complex. CONCLUSIONS. These results identify a novel regulatory mechanism for the hVps34/hVps15 complex in NT peptide secretion, providing a direct connection between AMPK and hVps34 which is independent of mTORC1/S6K1 signaling.