Type 2 diabetes is associated with insulin resistance and disturbances in pancreatic cell function that result in inadequate glucose-stimulated insulin secretion (GSIS). However, the mechanisms that cause cell failure are largely unknown. Recent studies implicate protein misfolding in the ER as a potential cause for cell failure in diabetic humans. Upon accumulation of unfolded proteins in the lumen of the ER, PERK, IREI?, and ATF6? 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 elF2?. Over the past cycle we demonstrated: 1) elF2? phosphorylation is required to limit protein synthesis and oxidative stress to maintain cell function; 2) the ER co-chaperone p58IPK is required to limit reactive oxygen species (ROS) and preserve cell function. Antioxidant treatment significantly restores cell function in p58IPK+/+ mice; and 3) IRE1?-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 cell is required to ensure the ER protein folding demand does not exceed the capacity. This is especially important for the 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 elF2? phosphorylation preserves cell function by limiting protein misfolding. We propose that excessive proinsulin synthesis causes proinsulin misfolding, ER Ca2+ 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 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 elF2?AA) cause cell dedifferentiation and can; antioxidants protect cells under these conditions? We will sort GFP+ elF2?AA cells from mice (+/- BHA-supplemented diet) and characterize their gene expression and D N A replication/damage patterns. b. Can reduced protein synthesis protect cells in elF2ccAA mice? We will test whether decreased protein synthesis through haploinsufficiency in the ribosomal protein RPL24 gene can protect elF2?AA cells. c. How does elF2? phosphorylation change 5' open reading frame (ORF) usage in mRNAs? Ribosomal protection assays will be performed to elucidate how elF2? phosphorylation alters ORF usage in response to glucose stimulation in wildtype and elF2?AA cells. d. Can pharmaceutical interventions protect elF2?AA cells that produce excessive proinsulin? We will test chemical chaperones, GLP-1, cyclosporin A, rapamycin/carbamazepine, etc. as a proof-of-concept that elF2?AA cell failure is due to protein misfolding and that agents known to improve ER protein folding will improve function of cells pressured by proinsulin synthesis.
Aim 2 : Proinsulin misfolding in the ER causes Ca2+ leak to mitochondria, leading to oxidative stress. a. Does p58IPKdeficiency 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 cell lines from p58IPK+/+ and p58IPK+/+ mice +/- glucose stimulation will be analyzed. b. Can SERCA overexpression improve insulin secretion and cell function in p58IPK+/+ cells and islets? c. Can cyclophilin D knockdown or deletion (Ppif-/-) prevent cell failure in p58IPK+/+ cells or mice, respectively? d. Can interventions in Id above improve function of p58IPK+/+ islets? For 2b-d, analyses will include methods described in 2a.
Aim 3 : IREI? and ATF6? 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 Ire1? change membrane association of mRNAs? b. Can antioxidants, cyclosporine A, or chemical chaperones improve ire1?-/- cell function and change mRNA cellular localization? c. Is Atfd? and/or Atf6 deletion detrimental to cells upon Ire1?-/- 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 #
4R37DK042394-20
Application #
9135961
Study Section
Special Emphasis Panel (NSS)
Program Officer
Haft, Carol R
Project Start
1998-01-01
Project End
2017-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
20
Fiscal Year
2016
Total Cost
$543,990
Indirect Cost
$260,044
Name
Sanford Burnham Prebys Medical Discovery Institute
Department
Type
DUNS #
020520466
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Zhang, Shuping; Macias-Garcia, Alejandra; Velazquez, Jason et al. (2018) HRI coordinates translation by eIF2?P and mTORC1 to mitigate ineffective erythropoiesis in mice during iron deficiency. Blood 131:450-461
Wang, Jie-Mei; Qiu, Yining; Yang, Zhao et al. (2018) IRE1? prevents hepatic steatosis by processing and promoting the degradation of select microRNAs. Sci Signal 11:
Poothong, Juthakorn; Tirasophon, Witoon; Kaufman, Randal J (2017) Functional analysis of the mammalian RNA ligase for IRE1 in the unfolded protein response. Biosci Rep 37:
Chiang, Wei-Chieh; Chan, Priscilla; Wissinger, Bernd et al. (2017) Achromatopsia mutations target sequential steps of ATF6 activation. Proc Natl Acad Sci U S A 114:400-405
Yao, Ting; Deng, Zhuo; Gao, Yong et al. (2017) Ire1? in Pomc Neurons Is Required for Thermogenesis and Glycemia. Diabetes 66:663-673
Choi, Woo-Gyun; Han, Jaeseok; Kim, Ji-Hyeon et al. (2017) eIF2? phosphorylation is required to prevent hepatocyte death and liver fibrosis in mice challenged with a high fructose diet. Nutr Metab (Lond) 14:48
DeZwaan-McCabe, Diane; Sheldon, Ryan D; Gorecki, Michelle C et al. (2017) ER Stress Inhibits Liver Fatty Acid Oxidation while Unmitigated Stress Leads to Anorexia-Induced Lipolysis and Both Liver and Kidney Steatosis. Cell Rep 19:1794-1806
Han, Jaeseok; Kaufman, Randal J (2017) Physiological/pathological ramifications of transcription factors in the unfolded protein response. Genes Dev 31:1417-1438
Poothong, Juthakorn; Sopha, Pattarawut; Kaufman, Randal J et al. (2017) IRE1? nucleotide sequence cleavage specificity in the unfolded protein response. FEBS Lett 591:406-414
Jin, Jung-Kang; Blackwood, Erik A; Azizi, Khalid et al. (2017) ATF6 Decreases Myocardial Ischemia/Reperfusion Damage and Links ER Stress and Oxidative Stress Signaling Pathways in the Heart. Circ Res 120:862-875

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