Type 2 diabetes is associated with insulin resistance and disturbances in pancreatic p cell function that result in inadequate glucose-stimulated insulin secretion (GSIS). However, the mechanisms that cause p cell failure are largely unknown. Recent studies implicate protein misfolding in the ER as a potential cause for p cell failure in diabetic humans. Upon accumulation of unfolded proteins in the lumen of the ER, PERK, IREIa, and ATF6a are activated to increase the capacity of the ER to meet the demand for increased protein folding and to increase the protein degradative machinery to eliminate misfolded proteins. In addition, protein synthesis is transiently attenuated through PERK-mediated phosphorylation of elF2a. Over the past cycle we demonstrated: 1) elF2a phosphorylation is required to limit protein synthesis and oxidative stress to maintain P cell function;2) the ER co-chaperone pSS""""""""""""""""*^ is required to limit reactive oxygen species (ROS) and preserve p cell function. Antioxidant treatment significantly restores p cell function in pSS'^*^'''mice;and 3) IRE1a-mediated splicing of Xbp1 mRNA induces co-transiational translocation at the ER to promote proinsulin production and represses oxidative stress. The sum of our data lead us to propose that tight control of protein synthesis in the p cell is required to ensure the ER protein folding demand does not exceed the capacity. This is especially important for the p cell as it is exposed to periodic postprandial increases in protein synthesis. In our Specific Aims, we will test three hypotheses by answering the following questions:
Aim 1 : Translational attenuation through elF2a phosphorylation preserves p cell function by limiting protein misfolding. We propose that excessive proinsulin synthesis causes proinsulin misfolding, ER Ca^* release and uptake into mitochondria, and mitochondrial-generated ROS. ROS then feed forward to further disrupt protein folding in the ER. Any stimuli that pressure p cells to exceed their capacity for proinsulin folding will succumb to this vicious cycle. To test this notion, we will answer: a. Does excessive,.(Proinsulin synthesis (such as elF2aAA) cause p cell dedifferentiation and can; antioxidants protect p cells under these conditions? We will sort GFP+ elF2aAA p cells from mice (+/- BHAsupplemented diet) and characterize their gene expression and D N A replication/damage patterns. b. Can reduced protein synthesis protect p cells in elF2ccAA mice? We will test whether decreased protein synthesis through haploinsufficiency in the ribosomal protein RPL24 gene can protect elF2aAA p cells. c. How does elF2a phosphorylation change 5'open reading frame (ORF) usage in mRNAs? Ribosomal protection assays will be performed to elucidate how elF2a phosphorylation alters ORF usage in response to glucose stimulation in wildtype and elF2aAA p cells. d. Can pharmaceutical interventions protect elF2aAA p cells that produce excessive proinsulin? We will test chemical chaperones, GLP-1, cyclosporin A, rapamycin/carbamazepine, etc. as a proof-of-concept that elF2aAA p cell failure is due to protein misfolding and that agents known to improve ER protein folding will improve function of p cells pressured by proinsulin synthesis.
Aim 2 : Proinsulin misfolding in the ER causes Ca^* leak to mitochondria, leading to oxidative stress. a. Does pSS""""""""""""""""*^ deficiency cause proinsulin misfolding in the ER to disrupt mitochondrial function and generate oxidative stress? Proinsulin synthesis, folding and trafficking, Ca^* imaging, mitochondrial membrane potential and ROS production in islets as well as in murine immortalized p cell lines from p58""""""""^'^*^* and p58""""""""''^'^'mice +/- glucose stimulation will be analyzed. b. Can SERCA overexpression improve insulin secretion and p cell function in p58""""""""''^""""""""^"""""""" cells and islets? c. Can cyclophilin D knockdown or deletion (Ppif^') prevent p cell failure in p58""""""""''^'''cells or mice, respectively? d. Can interventions in Id above improve function of pSS""""""""''^'''""""""""islets? For 2b-d, analyses will include methods described in 2a.
Aim 3 : IREIa and ATF6a provide overlapping functions to promote SRP-dependent ribosome and mRNA recruitment to the ER membrane during glucose stimulation and increase ER protein-folding capacity. a. How does Irel a change membrane association of mRNAs? b. Can antioxidants, cyclosporine A, or chemical chaperones improve ire1d''p cell function and change mRNA cellular localization? c. Is Atfda and/or Atfdp deletion detrimental to p cells upon I r e l d ''deletion, HFD feeding, or Akita mutation?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK042394-18
Application #
8730113
Study Section
No Study Section (in-house review) (NSS)
Program Officer
Haft, Carol R
Project Start
1998-01-01
Project End
2017-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
18
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Mollereau, B; Rzechorzek, N M; Roussel, B D et al. (2016) Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations. Brain Res 1648:603-616
Bai, Yongsheng; Kinne, Jeff; Donham, Brandon et al. (2016) Read-Split-Run: an improved bioinformatics pipeline for identification of genome-wide non-canonical spliced regions using RNA-Seq data. BMC Genomics 17 Suppl 7:503
Wang, Miao; Kaufman, Randal J (2016) Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 529:326-35
Ravindran, Rajesh; Loebbermann, Jens; Nakaya, Helder I et al. (2016) The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation. Nature 531:523-7
Han, Jaeseok; Kaufman, Randal J (2016) The role of ER stress in lipid metabolism and lipotoxicity. J Lipid Res 57:1329-38
Placzek, Andon N; Prisco, Gonzalo Viana Di; Khatiwada, Sanjeev et al. (2016) eIF2α-mediated translational control regulates the persistence of cocaine-induced LTP in midbrain dopamine neurons. Elife 5:
Yang, Liu; Li, Shaohua; Miao, Linqing et al. (2016) Rescue of Glaucomatous Neurodegeneration by Differentially Modulating Neuronal Endoplasmic Reticulum Stress Molecules. J Neurosci 36:5891-903
Kohl, Susanne; Zobor, Ditta; Chiang, Wei-Chieh et al. (2015) Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia. Nat Genet 47:757-65
Wang, Shiyu; Park, Shuin; Kodali, Vamsi K et al. (2015) Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100. Mol Biol Cell 26:594-604
Hassler, Justin R; Scheuner, Donalyn L; Wang, Shiyu et al. (2015) The IRE1α/XBP1s Pathway Is Essential for the Glucose Response and Protection of β Cells. PLoS Biol 13:e1002277

Showing the most recent 10 out of 100 publications