Down-regulation of the islet-specific zinc transporter-8 (ZnT8) protects human insulinoma cells against inflammatory stress

Chengfeng Merriman,Dax Fu

doi:10.1074/jbc.ra119.010937

Abstract

Zinc transporter-8 (ZnT8) primarily functions as a zinc-sequestrating transporter in the insulin-secretory granules (ISGs) of pancreatic β-cells. Loss-of-function mutations in ZnT8 are associated with protection against type-2 diabetes (T2D), but the protective mechanism is unclear. Here, we developed an in-cell ZnT8 assay to track endogenous ZnT8 responses to metabolic and inflammatory stresses applied to human insulinoma EndoC-βH1 cells. Unexpectedly, high glucose and free fatty acids did not alter cellular ZnT8 levels, but proinflammatory cytokines acutely, reversibly, and gradually down-regulated ZnT8. Approximately 50% of the cellular ZnT8 was localized to the endoplasmic reticulum (ER), which was the primary target of the cytokine-mediated ZnT8 down-regulation. Transcriptome profiling of cytokine-exposed β-cells revealed an adaptive unfolded protein response (UPR) including a marked immunoproteasome activation that coordinately degraded ZnT8 and insulin over a 1,000-fold cytokine concentration range. RNAi-mediated ZnT8 knockdown protected cells against cytokine cytotoxicity, whereas inhibiting immunoproteasomes blocked cytokine-induced ZnT8 degradation and triggered a transition of the adaptive UPR to cell apoptosis. Hence, cytokine-induced down-regulation of the ER ZnT8 level promotes adaptive UPR, acting as a protective mechanism that decongests the ER burden of ZnT8 to protect β-cells from proapoptotic UPR during chronic low-grade inflammation.

Highlights

Zinc transporter-8 (ZnT8) primarily functions as a zinc-sequestrating transporter in the insulin-secretory granules (ISGs) of pancreatic ␤-cells
Assay calibration showed a linear increase in the horseradish peroxidase (HRP) readout with a cell number that was proportional to the amount of endogenous ZnT8 (Fig. 1A)
Our results indicate that ZnT8 is temporally and spatially regulated to modulate zinc, endoplasmic reticulum (ER), and insulin homeostasis

Summary

To whom correspondence should be addressed

The onset of T2D and its progression are largely determined by a progressive failure of ␤-cells to produce sufficient amounts of insulin to compensate for insulin resistance. Excessive levels of glucose and FFA can induce local production and release of cytokines and chemokines from pancreatic islets, leading to macrophage recruitment and islet inflammation characterized by increased expression of inflammatory cytokines derived from innate immune cells [23]. Built on mAb20, an in-cell ELISA was developed to quantify fluctuations of the endogenous ZnT8 level in a multifactorial space of glucose (Glc), FFA, zinc, proinflammatory cytokines, and their time- and dose-dependent profiles. This precise assay revealed a highly sensitive ZnT8 response to cytokine stimulations. Our experiments revealed a novel immunologic process of decongesting two major ER burdens to protect ␤-cells from proapoptotic UPR

Results

Discussion

Experimental procedures