A metabolic redox relay supports ER proinsulin export in pancreatic islet β cells
Kristen E. Rohli, Nicole J. Stubbe, Emily M. Walker, Gemma L. Pearson, Scott A. Soleimanpour, Samuel B. Stephens
Kristen E. Rohli, Nicole J. Stubbe, Emily M. Walker, Gemma L. Pearson, Scott A. Soleimanpour, Samuel B. Stephens
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Research Article Cell biology Endocrinology

A metabolic redox relay supports ER proinsulin export in pancreatic islet β cells

Abstract

ER stress and proinsulin misfolding are heralded as contributing factors to β cell dysfunction in type 2 diabetes, yet how ER function becomes compromised is not well understood. Recent data identify altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple β cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that β cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas thioredoxin-interacting protein suppression restored ER redox and proinsulin trafficking. Taken together, we propose that β cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.

Authors

Kristen E. Rohli, Nicole J. Stubbe, Emily M. Walker, Gemma L. Pearson, Scott A. Soleimanpour, Samuel B. Stephens

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Figure 6

Idh1 suppression elicits ER hyperoxidation and impaired insulin granule formation.

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Idh1 suppression elicits ER hyperoxidation and impaired insulin granule...
Male and female 12- to 16-week-old mouse islets were treated with Ad-shSAFE or Ad-shIdh1 as indicated. (A) mRNA expression of Idh1 was determined by RT-qPCR (n = 5). NADPH/NADP+ (B, n = 4) and GSSG/GSH (C, n = 5–6) were measured in mCherry+ islet cells (Ad-shRNA) by sequential incubation in 2 mM Glc followed by 20 mM Glc for 12 minutes each via ratiometric imaging of iNAP or Grx1-roGFP2 (AdRIP), respectively. Responses were normalized to 2 mM Glc. (D) ER redox was measured in mCherry+ islet cells (Ad-shRNA; n = 4) via ratiometric imaging of ERroGFP (AdRIP). (E and F) ProCpepSNAP-expressing islets (AdRIP) were pulse-labeled with SNAP-505 (green) and chased for 2 hours. Cells were fixed and counterstained with DAPI (blue). The total number of nascent (proCpepSNAP-labeled) granules per mCherry+ cell was quantified (E, n = 4) and representative images are shown (F). (G and H) ProCpepSNAP-expressing islets (AdRIP) were cultured with vehicle (control) or DTT (0.5 mM) for 4 hours prior to pulse-labeling with SNAP-505 (green) and chased for 2 hours. Cells were fixed and counterstained with DAPI (blue). The total number of nascent (proCpepSNAP-labeled) granules per mCherry+ cell was quantified (G, n = 3–5) and representative images are shown (H). (A–E and G) Data represent the mean ± SEM. *P < 0.05 by 2-tailed Student’s t test (A–E) or 2-way ANOVA with Holm-Šídák posttest analysis (G). Scale bar = 5 μm.