An early hallmark of type 2 diabetes is a failure of proinsulin- to-insulin processing in pancreatic β-cells, resulting in hyperproinsulinemia. Proinsulin processing is quite sensitive to nutrient flux, and β-cell-specific deletion of the nutrientsensing protein modifier OGlcNAc transferase (βOGTKO) causes β-cell failure and diabetes, including early development of hyperproinsulinemia. The mechanisms underlying this latter defect are unknown. Here, using several approaches, including site-directed mutagenesis, Click O-GlcNAc labeling, immunoblotting, and immunofluorescence and EM imaging, we provide the first evidence for a relationship between the O-GlcNAcylation of eukaryotic translation initiation factor 4γ1 (eIF4G1) and carboxypeptidase E (CPE)-dependent proinsulin processing in βOGTKO mice. We first established that βOGTKO hyperproinsulinemia is independent of age, sex, glucose levels, and endoplasmic reticulum-CCAAT enhancerbinding protein homologous protein (CHOP)-mediated stress status. Of note, OGT loss was associated with a reduction in β-cell-resident CPE, and genetic reconstitution of CPE in βOGTKO islets rescued the dysfunctional proinsulin-to-insulin ratio. We show that although CPE is not directly OGlcNAc modified in islets, over expression of the suspected OGT target eIF4G1, previously shown to regulate CPE translation in-cells, increases islet CPE levels, and fully reverses βOGTKO islet-induced hyperproinsulinemia. Furthermore, our results reveal that OGT O-GlcNAc-modifies eIF4G1 at Ser-61 and that this modification is critical for eIF4G1 protein stability. Together, these results indicate a direct link between nutrient-sensitive OGT and insulin processing, underscoring the importance of post-translational O-GlcNAc modification in general cell physiology.
Bibliographical noteFunding Information:
This work was supported by National Institutes of Health, NIDDK Grants K01 DK103823, R21 DK112144, R03 DK114465, and R01 DK115720 (to E. U. A.), and F31 DK113694, 5T32DK083250 (to A. L). We acknowledge Regina Schlichting, Daniel Baumann, Dr. Ramkumar Mohan, and Ingrid Bender for technical support. We thank Drs. Peter Arvan, James Johnson, Ernesto Bernal- Mizrachi, David Bernlohr and Lauren Ball for discussion. We thank Dr. Thomas Pengo for his assistance in Fiji and the University of Minnesota Imaging Center for technical support. We thank Dr. Michael Kyba for access Electroporation and Nucleofector Device. The tissue processing and embedding was performed at the laboratory of Dr. Jop van Berlo.
This work was supported by National Institutes of Health, NIDDK Grants K01 DK103823, R21 DK112144, R03 DK114465, and R01 DK115720 (to E. U. A.), and F31 DK113694, 5T32DK083250 (to A. L). E. U.A. is the guarantor of this work. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
© 2019 Jo et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
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