Independent activation of CREB3L2 by glucose fills a regulatory gap in mouse -cells by co- ordinating insulin biosynthesis with secretory granule formation

Objective: Although individual steps have been characterized, there is little understanding of the overall process whereby glucose co-ordinates the biosynthesis of insulin with its export out of the endoplasmic reticulum (ER) and incorporation into insulin secretory granules (ISGs). Here we investigate a role for the transcription factor CREB3L2 in this context.Methods: MIN6 cells and mouse islets were analysed by immunoblotting after treatment with glucose, fatty acids, thapsigargin and various inhibitors. Knockdown of CREB3L2 was achieved using si or sh constructs by transfection, or viral delivery. In vivo metabolic phenotyping was conducted after deletion of CREB3L2 in beta-cells of adult mice using Ins1-CreER(+). Islets were isolated for RNAseq and assays of glucose-stimulated insulin secretion (GSIS). Trafficking was monitored in islet monolayers using a GFP-tagged proinsulin construct that allows for synchronised release from the ER.Results: With a K-m approximate to 3.5 mM, glucose rapidly (T-1/2 0.9 h) increased full length (FL) CREB3L2 followed by a slower rise (T-1/2 2.5 h) in its transcriptionally-active cleavage product, P60 CREB3L2. Glucose stimulation repressed the ER stress marker, CHOP, and this was partially reverted by knockdown of CREB3L2. Activation of CREB3L2 by glucose was not due to ER stress, however, but a combination of O-GlcNAcylation, which impaired proteasomal degradation of FL-CREB3L2, and mTORC1 stimulation, which enhanced its conversion to P60. cAMP generation also activated CREB3L2, but independently of glucose. Deletion of CREB3L2 inhibited GSIS ex vivo and, following a high-fat diet (HFD), impaired glucose tolerance and insulin secretion in vivo. RNAseq revealed that CREB3L2 regulated genes controlling trafficking to-and-from the Golgi, as well as a broader cohort associated with beta-cell compensation during a HFD. Although post-Golgi trafficking appeared intact, knockdown of CREB3L2 impaired the generation of both nascent ISGs and proinsulin condensates in the Golgi, implying a defect in ER export of proinsulin and/or its processing in the Golgi.Conclusion: The stimulation of CREB3L2 by glucose defines a novel, rapid and direct mechanism for co-ordinating the synthesis, packaging and storage of insulin, thereby minimizing ER overload and optimizing beta-cell function under conditions of high secretory demand. Upregulation of CREB3L2 also potentially contributes to the benefits of GLP1 agonism and might in itself constitute a novel means of treating beta-cell failure.Crown Copyright (c) 2023 Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).