Requirement For Translocon-associated Protein Alpha (TRAPα) In Insulin Biogenesis

The discovery of missense mutations in the signal peptide of preproinsulin that causes congenital diabetes highlights the critical role of ER translocation in insulin biosynthesis.However,the mechanisms that govern these early events in insulin biosynthesis are poorly understood.GWAS studies have shown that transloconassociated protein alpha(TRAPα)is a high-risk allele for both type 2 diabetes and gestational diabetes.Yet,the pathophysiological role of TRAPα in β-cell function remains unknown.Here we show that TRAPα is the most upregulated subunit of the TRAP complexes in isolated islets exposed to high glucose,suggesting that it may play an important role in insulin biosynthesis.CRISPR/Cas9 mediated TRAPα deletion in pancreatic INS832/13 cells caused inefficient preproinsulin translocation across the ER membrane,resulted in an accumulation of un-translocated preproinsulin in the cells.Surprisingly,TRAPα deletion also affects proinsulin ER export,leading to a decreased insulin production in INS832/13 cells.Adenoviral re-introducing TRAPα to TRAPα KO cells rescued preproinsulin translocation defect and restored insulin production in a dose-dependent manner.Importantly,TRAPα deletion did not significantly affect the biogenesis or secretion of α1-antitrypsin,indicating that TRAPα promotes the ER translocation and subsequent trafficking of only a subset of secreted proteins.Targeted disruption of TRAPα in mouse pancreatic β-cells had no significant effects on the mouse body weight,fasting blood glucose,and fasting insulin in both regular chow and high-fat diet(HFD)fed mice.However,IPGTT revealed that glucose tolerance and insulin secretion response to glucose were significantly impaired in the TRAPα βKO mice fed with regular chow,and these impairments were further aggravated in HFD fed mice.Consistent with these phenotypes,glucose-stimulated insulin secretion(GSIS)was markedly impaired in isolated islets from TRAPα βKO mice.Although insulin content was decreased,neither islet morphology nor cell composition was significantly affected by TRAPα βKO.Consistent with the role of TRAPα in insulin biosynthesis in β-cell lines,we found that preproinsulin translocation was impaired along with markedly decreased intracellular mature insulin in the islets isolated from TRAPα βKO mice,supporting the notion that TRAPα is critical for maintaining β-cell function in vivo.These results establish preproinsulin as a client protein for TRAPα in ER translocation and unveil unanticipated function of TRAPα in proinsulin ER export and insulin biosynthesis.Our study also provides the first in vivo evidence that ablation of TRAPα leads to defects in insulin production and glucose intolerance,and illustrates key insights into the disease pathogenesis associated with TRAPα deficiency.