ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet β-Cell

Abstract

Defects in the pancreatic β-cell's secretion system are well-described in Type 2 diabetes (T2D) and include impaired proinsulin processing and a deficit in mature insulin-containing secretory granules; however, the cellular mechanisms underlying these defects remain poorly understood. To address this, we used an in situ fluorescent pulse-chase strategy to study proinsulin trafficking. We show that insulin granule formation and the appearance of nascent granules at the plasma membrane are decreased in rodent and cell culture models of pre-diabetes and hyperglycemia. Moreover, we link the defect in insulin granule formation to an early trafficking delay in ER export of proinsulin, which is independent of overt ER stress. Using a ratiometric redox sensor, we show that the ER becomes hyperoxidized in β-cells from a dietary model of rodent pre-diabetes and that addition of reducing equivalents restores ER export of proinsulin and insulin granule formation and partially restores β-cell function. Together, these data identify a critical role for the regulation of ER redox homeostasis in proinsulin trafficking and suggest that alterations in ER redox poise directly contribute to the decline in insulin granule production in T2D.

Synchronized proinsulin trafficking reveals delayed Golgi export accompanies β-cell secretory dysfunction in a rodent model of hyperglycemia

“…Despite the strong association of ER stress with the onset of β-cell dysfunction 47 , morphological changes to ER structure are relatively modest in T2D and largely include expansion of the ER membrane surface, rather than dilation of the ER lumen 12,45,46 . ER redox imbalance and proinsulin misfolding in T2D can lead to the formation of disulfide-linked aggregates in the ER [10][11][12] . Presumably, ER-associated degradation (ERAD) and ER autophagy are utilized to eliminate some aggregates [48][49][50] ; however, a portion of misfolded proinsulin may exit the ER and accumulate in the Golgi leading to the swollen cisternae observed here.…”

“…We used proCpepRUSH granule distance from the Golgi to assess trafficking and normalized the data as a relative frequency of granule distances per cell imaged, which accounts for variations in reporter expression between cells and conditions 12,19 . As shown in Figure 4E, proCpepRUSHpositive granules (green) in β-cells from SC fed mice were distributed throughout the cell body.…”

“…ProCpepRUSH (SA-KDEL-IRES-proCpep-SBP-GFP) was generated by Gibson assembly and subcloned into pENTR2b-RIP 12 . SA-KDEL-IRES and SBP PCR fragments were separately amplified from Str-KDEL_SBP-EGFP-GPI (gift from Franck Perez, Addgene plasmid #65294; RRID:Addgene_65294) 28 .…”

“…The copyright holder for this preprint this version posted November 1, 2022. ; https://doi.org/10.1101/2022.10.31.514578 doi: bioRxiv preprint from spot-rendered proCpepRUSH-positive granules (150-300 nm) and surface rendering of the Golgi identified through GM130 immunostaining. Granule distances were binned as indicated and expressed as a percentage of the total to normalize between cells and conditions 12,19 .…”

“…From the initial synthesis of proinsulin, to the endoproteolytic conversion to insulin in the maturing granule, pulse-chase radiolabeling studies estimate insulin biosynthesis requires approximately 3 hours 5 . Proinsulin folding in the endoplasmic reticulum (ER) is widely regarded as a rate-limiting step in the secretory biosynthetic pathway and recent data highlights that impairments in proinsulin folding and disulfide bond formation limit insulin granule formation in diabetes models [10][11][12] . In addition, proinsulin exit from the trans-Golgi network (TGN) may also be a key regulated step in the insulin biosynthetic pathway 13,14 .…”