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bioRxiv · 10.1101/2024.12.26.629558

Target deconvolution of an insulin hypersecretion-inducer acting through VDAC1 with a distinct transcriptomic signature in beta-cells

Roy, G.·Ordonez, A.·Binns, D. D.·Rodriguez-dos-Santos, K.·Kwakye, M. B.·King, G. C.·Mukherjee, N.·Templin, A. T.·Tan, Z.-y.·Richardson, T. I.·Doud, E. H.·Mosley, A. L.·Emfinger, C. H.·Attie, A. D.·Keller, M. P.·Johnson, T. S.·Kalwat, M. A.
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Abstract

Obesity, insulin resistance, and a host of environmental and genetic factors can drive hyperglycemia, causing {beta}-cells to compensate by increasing insulin production and secretion. In type 2 diabetes (T2D), {beta}-cells under these conditions eventually fail. Rare {beta}-cell diseases like congenital hyperinsulinism (HI) also cause inappropriate insulin secretion, and some HI patients develop diabetes. However, the mechanisms of insulin hypersecretion and how it causes {beta}-cell dysfunction are not fully understood. We previously discovered small molecules (e.g. SW016789) that cause insulin hypersecretion and lead to a loss in {beta}-cell function without cell death. Here, we uncover the protein target of SW016789 and provide the first time-course transcriptomic analysis of hypersecretory responses versus thapsigargin-mediated ER stress in {beta}-cells. In mouse MIN6 and human EndoC-{beta}H1 {beta}-cells, we identified and validated VDAC1 as a SW016789 target using photoaffinity proteomics, cellular thermal shift assays, siRNA, and small molecule inhibitors. SW016789 raises membrane potential to enhance Ca2+ influx, potentially through VDAC1. Chronically elevated intracellular Ca2+ appears to underpin the negative impacts of hypersecretion, as nifedipine protected against each small molecule hypersecretion inducer we tested. Using time- course RNAseq, we discovered that hypersecretion induced a distinct transcriptional pattern compared to ER stress. Clustering analyses led us to focus on ER-associated degradation (ERAD) as a potential mediator of the adaptive response. SW016789 reduced the abundance of ERAD substrate OS-9 and pharmacological inhibition of ERAD worsened {beta}-cell survival in response to hypersecretory stress. Changes in other ERAD components in MIN6 and EndoC-{beta}H1 at the protein level were minor with either SW016789 or thapsigargin. However, immunostaining for core ERAD components SEL1L, HRD1, and DERL3 in non-diabetic and T2D human pancreas revealed altered distributions of SEL1L/HRD1 and SEL1L/DERL3 rations in {beta}-cells of T2D islets, in alignment with altered ERAD in stressed {beta}-cells. We conclude that hypersecretory stimuli, including SW016789- mediated VDAC1 activation, cause enhanced Ca2+ influx and insulin release. Subsequent differential gene expression represents a {beta}-cell hypersecretory response signature that is reflected at the protein level for some, but not all genes. A better understanding of how {beta}-cells induce hypersecretion and the mechanisms of negative feedback on secretory rate may lead to the discovery of novel therapeutic targets for T2D and HI.

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Roy, G., Ordonez, A., Binns, D. D., Rodriguez-dos-Santos, K., Kwakye, M. B., King, G. C., Mukherjee, N., Templin, A. T., Tan, Z.-y., Richardson, T. I., Doud, E. H., Mosley, A. L., Emfinger, C. H., Attie, A. D., Keller, M. P., Johnson, T. S., Kalwat, M. A.. 2024-12-26. Target deconvolution of an insulin hypersecretion-inducer acting through VDAC1 with a distinct transcriptomic signature in beta-cells. https://doi.org/10.1101/2024.12.26.629558

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