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 1

β Cell dysfunction and ER hyperoxidation coincide with impaired mitochondrial function and diminished cellular redox.

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β Cell dysfunction and ER hyperoxidation coincide with impaired mitochon...
Human islets (A–C) or INS-1 832/3 cells (D–I) were cultured for 3 days in control media supplemented with BSA (5.5 mM or 11 mM Glc, respectively) or media supplemented with oleate/palmitate (2:1, 1 mM) and elevated glucose (20 mM), labeled OPG, as indicated. (A and D) Insulin secretion was measured by static incubation in media containing 2.5 mM Glc followed by 12 mM Glc for 1 hour each (n = 5 human islet donors; n = 5 INS-1 experiments). (B) Insulin content was measured from human islet cell lysates (n = 5). (C and E) ER redox was determined from ratiometric imaging of cells expressing ERroGFP (AdRIP) (n = 4 or n = 5, respectively). (F) Oxygen consumption rate was measured following sequential addition of oligomycin A (2.5 μM), FCCP (1.25 μM), and rotenone plus antimycin A (2 μM each) as indicated (n = 3). (G) Cells were cocultured with MitoQ (0.5 μM) as indicated. ER redox was determined from ratiometric imaging of cells expressing ERroGFP (AdRIP) (n = 3). NADPH/NADP+ (H) or GSSG/GSH (I) were measured by sequential incubation in 2 mM Glc followed by 20 mM Glc for 12 minutes each via ratiometric imaging of iNAP (n = 4) or Grx1-roGFP2 (n = 7–8) (AdRIP), respectively. Responses were normalized to 2 mM Glc. (A–I) Data represent the mean ± SEM. *P < 0.05 by 2-way ANOVA with Tukey’s posttest analysis (A and G), 2-way ANOVA with Holm-Šídák posttest analysis (D), or 2-tailed Student’s t test (B, C, E, H, and I).