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

Abstract

Edited by Jeffrey E. Pessin Zinc transporter-8 (ZnT8) primarily functions as a zinc-sequestrating transporter in the insulin-secretory granules (ISGs) of pancreatic ␤-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 incell ZnT8 assay to track endogenous ZnT8 responses to metabolic and inflammatory stresses applied to human insulinoma EndoC-␤H1 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 ␤-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. RNAimediated 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 ␤-cells from proapoptotic UPR during chronic low-grade inflammation.

Zinc transporters and their functional integration in mammalian cells

“…The resultant cytosolic zinc increase was proposed to inhibit protein–tyrosine phosphatase 1B (PTP1B) activity, which acts to suppress apoptosis and steatosis associated with hepatic ER stress. Likewise in human EndoC-βH1 pancreatic β cells, cytokine-induced ER stress also significantly upregulated both ZIP7 and ZIP14 expression ( 170 ), consistent with protective roles of both ZIP7 and ZIP14 in promoting adaptive UPRs in diverse mammalian cell types.…”

“…It functions as a zinc gatekeeper that tightly controls the movement of zinc from the ER zinc store into the cytosol and relies on phosphorylation to activate its zinc transport activity ( 113 ). In pancreatic β cells, ZIP7 is the most abundantly expressed among ZIP homologs ( 170 ), and its mRNA level is upregulated in response to glucose stimulation ( 171 ). Functionally, ZIP7 and the surface-expressed ZIP6 are mutually compensatory in β cells.…”

“…ZnT8 is a major zinc-sequestrating transporter responsible for zinc enrichment in ISGs ( 148 , 149 ) and insulin packaging ( 150 , 202 , 203 , 204 , 205 ). Accordingly, ZnT8 expression in β-cells is over ten times more abundant than other zinc transporters at the mRNA level ( 206 , 207 ), and its protein level in EndoC-βH1 cells is compatible to that of the housekeeping α-tubulin ( 170 ). Importantly, the subcellular localizations of ZnT8 have an even distribution between the ER and ISGs, contributing significantly to both zinc loading and protein folding in the ER ( 170 ).…”

“…Surprisingly, the ZnT8 protein level in human EndoC-βH1 cells remained unchanged in the face of large fluctuations of insulin synthesis and secretion upon physiological glucose stimulations ( 170 ). In contrast, a trace amount of proinflammatory cytokines triggered a rapid, graded, and reversible degradation of ER-resident ZnT8 while the ISG-resident ZnT8 remained largely unchanged ( 170 ). These findings demonstrated exquisite specificity of ER-resident ZnT8 to cytokine stimulations.…”

ZnT8 Haploinsufficiency Impacts MIN6 Cell Zinc Content and β-Cell Phenotype via ZIP-ZnT8 Coregulation

“…MAPK cascades are activated by physiological concentrations of Zn 2+ [54,55] to regulate gene expression, mitosis, differentiation, metabolism and programmed cell death [56]. The protective effects of reduced ZnT8 expression in vivo may be due to the ability to protect against cytokine cytotoxicity [57], which was not investigated in this study. Interestingly, the two zinc importers that showed changed expression in ZnT8 haploinsufficient cells were ZIP8 and ZIP14, which both have functions in the immune response [58,59].…”