The peptide hormone, insulin, regulates metabolism to homeostatically maintain blood glucose levels within a narrow physiological range. In pancreatic ss-cells, insulin is made and stored at high concentration within secretory granules. Physiological stimulation of insulin secretion (multiple times per day) requires very active synthesis of new insulin to replenish secretory granule reserves. Insulin synthesis begins with translation of preproinsulin for delivery into the lumen of the endoplasmic reticulum (ER). Therein, proinsulin must properly fold, which is easier than it sounds: proinsulin is a "disulfide-challenged" protein. Moreover, when beta cells are forced to synthesize higher levels of proinsulin than they are genetically-programmed to handle, they risk further proinsulin misfolding/disulfide mispairing, which leads to secretory pathway stress. The objective of this new grant cycle is to better understand proinsulin folding and export from the ER. We hypothesize that misfolding of a subfraction of proinsulin in the ER can block insulin production derived from the other subfracton of "bystander" proinsulin molecules, backlogging the protein in the ER, and driving ER stress, beta cell failure, loss of pancreatic insulin content, and diabetes. We propose four Specific Aims: 1) To elucidate the molecular mechanism(s) by which newly-described point mutations in the coding sequence of preproinsulin lead to human diabetes in neonates and adults. 2) To characterize ss-cell ER oxidoreductases. 3) To develop a new cell culture-based system to dissect steps leading to beta cell death. 4) To develop an in vivo analysis of pancreatic insulin production in diabetes.

Public Health Relevance

Insulin regulates metabolism to maintain normal blood glucose levels. Synthesis of new insulin begins with translation of proinsulin in the endoplasmic reticulum. In the past year, more than 20 new proinsulin mutations have been found to be associated with neonatal onset diabetes. Evidence suggests that these mutant proinsulins are made as proteins but it is not known how they cause diabetes. Each patient also havs another allele of perfectly normal proinsulin which should be more than enough proinsulin synthesis to satisfy the body's need for insulin. This new grant cycle proposes experiments to better understand proinsulin folding and export in order to see how misfolding of a subfraction of proinsulin in the ER can block insulin production derived from "bystander" proinsulin molecules, causing ER stress, beta cell failure, and diabetes.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Haft, Carol R
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University of Michigan Ann Arbor
Internal Medicine/Medicine
Schools of Medicine
Ann Arbor
United States
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Liu, Ming; Wright, Jordan; Guo, Huan et al. (2014) Proinsulin entry and transit through the endoplasmic reticulum in pancreatic beta cells. Vitam Horm 95:35-62
Prabhu, Yogikala; Blanco, Elias H; Liu, Ming et al. (2014) Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function. Endocrinology 155:2391-401
Guo, Huan; Xiong, Yi; Witkowski, Piotr et al. (2014) Inefficient translocation of preproinsulin contributes to pancreatic ? cell failure and late-onset diabetes. J Biol Chem 289:16290-302
Haataja, Leena; Snapp, Erik; Wright, Jordan et al. (2013) Proinsulin intermolecular interactions during secretory trafficking in pancreatic ? cells. J Biol Chem 288:1896-906
Wright, Jordan; Birk, Julia; Haataja, Leena et al. (2013) Endoplasmic reticulum oxidoreductin-1? (Ero1?) improves folding and secretion of mutant proinsulin and limits mutant proinsulin-induced endoplasmic reticulum stress. J Biol Chem 288:31010-8
Liu, Ming; Lara-Lemus, Roberto; Shan, Shu-ou et al. (2012) Impaired cleavage of preproinsulin signal peptide linked to autosomal-dominant diabetes. Diabetes 61:828-37
Rajpal, Gautam; Schuiki, Irmgard; Liu, Ming et al. (2012) Action of protein disulfide isomerase on proinsulin exit from endoplasmic reticulum of pancreatic ?-cells. J Biol Chem 287:43-7
Hodish, Israel; Absood, Afaf; Liu, Leanza et al. (2011) In vivo misfolding of proinsulin below the threshold of frank diabetes. Diabetes 60:2092-101
Khoo, Cynthia; Yang, Juxiang; Rajpal, Gautam et al. (2011) Endoplasmic reticulum oxidoreductin-1-like * (ERO1l*) regulates susceptibility to endoplasmic reticulum stress and is induced by insulin flux in *-cells. Endocrinology 152:2599-608
Liu, Ming; Haataja, Leena; Wright, Jordan et al. (2010) Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport. PLoS One 5:e13333

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