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Insulin Regulates Carboxypeptidase E by Modulating Translation Initiation

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Insulin Regulates Carboxypeptidase E by Modulating Translation Initiation Insulin regulates carboxypeptidase E by modulating PNAS PLUS translation initiation scaffolding protein eIF4G1 in pancreatic β cells Chong Wee Liewa,b,1,2, Anke Assmanna,1, Andrew T. Templinc, Jeffrey C. Raumd, Kathryn L. Lipsone,3, Sindhu Rajanf, Guifen Qiangb, Jiang Hua, Dan Kawamoria, Iris Lindbergg, Louis H. Philipsonf, Nahum Sonenbergh, Allison B. Goldfinea, Doris A. Stoffersd, Raghavendra G. Mirmirac, Fumihiko Uranoi, and Rohit N. Kulkarnia,2 aJoslin Diabetes Center and Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02215; bDepartment of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612; cDepartment of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202; dDepartment of Medicine/Endocrinology, Diabetes, and Metabolism and the Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104; eProgram in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605; fDepartment of Medicine, The University of Chicago, Chicago, IL 60637; gDepartment of Anatomy and Neurobiology, University of Maryland, Baltimore, MD 21201; hDepartment of Biochemistry, McGill University, Montreal, QC, Canada H3A 1A3; and iDivision of Endocrinology, Metabolism, and Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110 Edited by David J. Mangelsdorf, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 29, 2014 (received for review December 24, 2013) Insulin resistance, hyperinsulinemia, and hyperproinsulinemia oc- modification of the properly folded proinsulin in the secretory cur early in the pathogenesis of type 2 diabetes (T2D). Elevated granules by prohormone convertase (PC) 1/3, PC2, and car- levels of proinsulin and proinsulin intermediates are markers of boxypeptidase E (CPE) (12–16). However, the effects of insulin β-cell dysfunction and are strongly associated with development signaling on posttranslational processing of insulin are not fully of T2D in humans. However, the mechanism(s) underlying β-cell explored. dysfunction leading to hyperproinsulinemia is poorly understood. In addition to insulin’s actions in classical insulin-responsive Here, we show that disruption of insulin receptor (IR) expression in tissues (muscle, liver, and fat), insulin signaling regulates β-cell β cells has a direct impact on the expression of the convertase mass and function (17–22), as well as transcription of the insu- enzyme carboxypeptidase E (CPE) by inhibition of the eukaryotic lin gene itself (23). We hypothesized that disruption of normal translation initiation factor 4 gamma 1 translation initiation com- growth factor (insulin) signaling in the β cell has an impact on plex scaffolding protein that is mediated by the key transcription proinsulin processing and/or adversely affects the function of the factors pancreatic and duodenal homeobox 1 and sterol regulatory ER and, ultimately, the β cell. In this study, to examine whether element-binding protein 1, together leading to poor proinsulin disruption of the insulin-signaling pathway has a direct impact on processing. Reexpression of IR or restoring CPE expression each proinsulin content, we examined the pancreas and islets from independently reverses the phenotype. Our results reveal the identity of key players that establish a previously unknown link Significance between insulin signaling, translation initiation, and proinsulin processing, and provide previously unidentified mechanistic in- Elevated circulating proinsulin and a poor biological response sight into the development of hyperproinsulinemia in insulin- to insulin are observed early in individuals with type 2 di- resistant states. abetes. Genome-wide association studies have recently iden- tified genes associated with proinsulin processing, and clinical ER stress | prohormone | bIRKO | GWAS observations suggest that elevated proinsulin and its inter- mediates are markers of dysfunctional insulin-secreting β cells. levated proinsulin and processing intermediates in the cir- Here, we propose a previously unidentified mechanism in the Eculation of patients with type 2 diabetes (T2D) suggest a regulation of an enzyme that is involved in proinsulin pro- defect in hormone processing in β-cell secretory granules (1–3). cessing called carboxypeptidase E (CPE). Disruption of insulin Genome-wide association studies have recently identified several signaling in β cells reduces expression of a scaffolding protein, SNPs in or near the TCF7L2, SLC30A8, SGSM2, VPS13C, eukaryotic translation initiation factor 4 gamma 1, that is re- MAPK-activating death domain (MADD), and ADCY5 genes quired for the initiation of translation and occurs via regulation that are associated with either altered proinsulin levels or pro- of two transcription factors, namely, pancreatic and duodenal insulin-to-insulin conversion (4–6). These findings gain signifi- homeobox 1 and sterol regulatory element-binding protein 1. cance because an increase in the proinsulin-to-insulin ratio Together, these effects lead to reduced levels of CPE protein predicts future development of T2D in apparently healthy indi- and poor proinsulin processing in β cells. viduals (7, 8). Given that proinsulin has only ∼5% of the biological activity of mature insulin, an increase in circulating Author contributions: C.W.L., A.A., A.B.G., D.A.S., R.G.M., F.U., and R.N.K. designed re- proinsulin is predicted to limit the actions of mature insulin and, search; C.W.L., A.A., A.T.T., J.C.R., K.L.L., S.R., G.Q., J.H., D.K., and I.L. performed research; I.L., L.H.P., N.S., D.A.S., and R.G.M. contributed new reagents/analytic tools; C.W.L., A.A., consequently, to contribute to worsening glucose tolerance in A.T.T., J.C.R., K.L.L., S.R., D.K., D.A.S., R.G.M., F.U., and R.N.K. analyzed data; and C.W.L., humans (9). Other studies have reported increased circulating A.A., and R.N.K. wrote the paper. proinsulin in insulin-resistant obese subjects with normal glucose The authors declare no conflict of interest. MEDICAL SCIENCES tolerance compared with nonobese individuals (10, 11), sug- This article is a PNAS Direct Submission. gesting a potential role for insulin resistance in proinsulin pro- 1C.W.L. and A.A. contributed equally to this work. cessing. However, the precise molecular mechanisms underlying 2 β To whom correspondence may be addressed. E-mail: [email protected] -cell dysfunction that promote hyperproinsulinemia remain poorly or [email protected]. understood. 3Present address: Department of Physical and Biological Sciences, Western New England The biosynthesis of insulin is regulated at multiple levels, in- University, Springfield, MA 01119. cluding transcription as well as posttranslational protein folding This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. at the endoplasmic reticulum (ER) and proteolytic cleavage and 1073/pnas.1323066111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1323066111 PNAS | Published online May 19, 2014 | E2319–E2328 Downloaded by guest on October 1, 2021 mice with insulin receptor knockout in the β cells (βIRKO), a mouse model manifesting a phenotype that resembles human T2D (19), and we also investigated β-cell lines lacking the insulin receptor (IR) (20). We have previously reported that βIRKO mice developed age-dependent, late-onset T2D (19) with an in- crease in the ratio of circulating total insulin to C-peptide suggesting elevated proinsulin secretion by βIRKO cells. However, the potential contribution of proinsulin in the development of T2D remains unknown. We demonstrate an increased accumulation of proinsulin in the βIRKO cells due to altered expression of PC enzymes, es- pecially CPE. These changes are mediated by duodenal ho- meobox protein (Pdx1) and sterol regulatory element-binding protein 1 (SREBP1) transcriptional regulation of the translation initiation complex scaffolding protein, eukaryotic translation initiation factor 4 gamma (eIF4G) 1, and indicate a previously unidentified role for these transcription factors in the regulation of translational initiation. Reexpression of the IR in the βIRKO cells, knocking down proinsulin, or maintaining normal expres- sion of CPE each independently restores the normal phenotype in mutant β cells. Together, these data point to previously un- identified links between insulin signaling, translational initiation, and proinsulin processing. Results Lack of IRs in β Cells Promotes Proinsulin Accumulation. To in- vestigate the role of proinsulin in the development of diabetes in βIRKO mice, we performed longitudinal studies in control and βIRKO male mice fed a chow diet from the age of 2–7 mo. We observed that both control and βIRKO mice at the age of 4 mo exhibited an increase in the proinsulin/insulin ratio compared Fig. 1. Increase in proinsulin due to down-regulation of CPE in β cells with their respective levels at 2 mo, despite unaltered fed blood lacking IRs. (A) Blood glucose and circulating proinsulin-to-insulin ratio in glucose levels (Fig. 1A), and argue against the primary role of control (Con) or βIRKO mice aged 2–7mo(n = 5–9). (B) Immunostaining and glucose in the modulation of proinsulin levels. Interestingly, we quantification for proinsulin (green), insulin (red), and DAPI (blue) in pan- also observed that βIRKO mice manifested a much greater el- creas
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