title: Substrate-favored lysosomal and proteasomal pathways participate in the normal balance control of insulin precursor maturation and disposal in β-cells.
abstract: Our recent studies have uncovered that aggregation-prone proinsulin preserves a low relative folding rate and maintains a homeostatic balance of natively and non-natively folded states (i.e., proinsulin homeostasis, PIHO) in β-cells as a result of the integration of maturation and disposal processes. Control of precursor maturation and disposal is thus an early regulative mechanism in the insulin production of β-cells. Herein, we show pathways involved in the disposal of endogenous proinsulin at the early secretory pathway. We conducted metabolic-labeling, immunoblotting, and immunohistochemistry studies to examine the effects of selective proteasome and lysosome or autophagy inhibitors on the kinetics of proinsulin and control proteins in various post-translational courses. Our metabolic-labeling studies found that the main lysosomal and ancillary proteasomal pathways participate in the heavy clearance of insulin precursor in mouse islets/β-cells cultured at the mimic physiological glucose concentrations. Further immunoblotting and immunohistochemistry studies in cloned β-cells validated that among secretory proteins, insulin precursor is heavily and preferentially removed. The rapid disposal of a large amount of insulin precursor after translation is achieved mainly through lysosomal autophagy and the subsequent basal disposals are carried out by both lysosomal and proteasomal pathways within a 30 to 60-minute post-translational process. The findings provide the first clear demonstration that lysosomal and proteasomal pathways both play roles in the normal maintenance of PIHO for insulin production, and defined the physiological participation of lysosomal autophagy in the protein quality control at the early secretory pathway of pancreatic β-cells.
authors.affiliation: Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Zhang
authors.forename: Qingxin
authors.initials: Q
authors.lastname: Yuan
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.forename: Jingyu
authors.initials: J
authors.lastname: Gu
authors.forename: Kwame
authors.initials: K
authors.lastname: Osei
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D001343:Autophagy; D015153:Blotting, Western; D005947:Glucose / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D011384:Proinsulin / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D014176:Protein Biosynthesis
chemical_list: D007328:Insulin; D011384:Proinsulin; D046988:Proteasome Endopeptidase Complex; D005947:Glucose
references.citation: Nature. 1969 Jan 11;221(5176):188-90
references.pmid: 5782717
references.citation: J Cell Biol. 1994 Jul;126(1):77-86
references.pmid: 8027188
references.citation: Cell. 2006 Aug 25;126(4):727-39
references.pmid: 16923392
references.citation: Nature. 2009 Mar 26;458(7237):453-60
references.pmid: 19325625
references.citation: Cell. 2008 Jan 11;132(1):27-42
references.pmid: 18191218
references.citation: J Biol Chem. 2006 May 12;281(19):13015-13020
references.pmid: 16543239
references.citation: J Biol Chem. 1995 Sep 1;270(35):20417-23
references.pmid: 7657617
references.citation: Cell Metab. 2008 Oct;8(4):325-32
references.pmid: 18840363
references.citation: Methods Enzymol. 2009;453:325-42
references.pmid: 19216914
references.citation: J Biol Chem. 2009 Oct 2;284(40):27664-73
references.pmid: 19654319
references.citation: J Cell Biol. 1984 Jan;98(1):222-8
references.pmid: 6368567
references.citation: EMBO J. 2000 Nov 1;19(21):5720-8
references.pmid: 11060023
references.citation: Trends Cell Biol. 2004 Feb;14(2):70-7
references.pmid: 15102438
references.citation: Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327-31
references.pmid: 28524
references.citation: Trends Cell Biol. 2007 Jun;17(6):279-85
references.pmid: 17481899
references.citation: PLoS One. 2011 Apr 29;6(4):e19446
references.pmid: 21559376
references.citation: Nature. 2007 Nov 29;450(7170):663-9
references.pmid: 18046402
references.citation: J Biol Chem. 1991 Jul 25;266(21):13737-41
references.pmid: 1856207
references.citation: J Biol Chem. 1979 May 10;254(9):3483-92
references.pmid: 372192
references.citation: Science. 1973 Jul 20;181(4096):223-30
references.pmid: 4124164
references.citation: Proc Natl Acad Sci U S A. 1978 Mar;75(3):1260-4
references.pmid: 206890
references.citation: Diabetes. 2006 Aug;55(8):2192-201
references.pmid: 16873681
references.citation: Proc Natl Acad Sci U S A. 1968 Jun;60(2):622-9
references.pmid: 5248819
references.citation: J Biol Chem. 1990 May 5;265(13):7478-84
references.pmid: 2185250
references.citation: J Cell Biol. 1998 Sep 7;142(5):1313-24
references.pmid: 9732291
references.citation: EMBO J. 2008 Jan 23;27(2):315-27
references.pmid: 18216874
references.citation: Curr Opin Cell Biol. 2009 Aug;21(4):516-21
references.pmid: 19443192
references.citation: J Biol Chem. 1983 Oct 10;258(19):11487-91
references.pmid: 6352698
references.citation: Semin Cell Dev Biol. 2000 Aug;11(4):235-42
references.pmid: 10966857
references.citation: Diabetes. 2008 Apr;57(4):1034-42
references.pmid: 18162506
references.citation: Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11089-93
references.pmid: 8248213
references.citation: Cell Metab. 2008 Oct;8(4):318-24
references.pmid: 18840362
references.citation: Proc Natl Acad Sci U S A. 2007 Sep 18;104(38):15040-4
references.pmid: 17855560
references.citation: Genes Cells. 2004 Mar;9(3):261-70
references.pmid: 15005713
references.citation: Nature. 2000 Apr 13;404(6779):770-4
references.pmid: 10783891
references.citation: J Biol Chem. 2005 Apr 22;280(16):15727-34
references.pmid: 15705591
references.citation: Proc Natl Acad Sci U S A. 1982 Mar;79(6):1889-92
references.pmid: 6952238
references.citation: J Clin Invest. 1999 Jan;103(1):27-37
references.pmid: 9884331
references.citation: Autophagy. 2007 May-Jun;3(3):285-7
references.pmid: 17351330
references.citation: J Cell Biol. 2008 Jul 28;182(2):225-32
references.pmid: 18644895
references.citation: Endocr Rev. 2008 May;29(3):317-33
references.pmid: 18436705
references.citation: J Biol Chem. 2003 Jul 4;278(27):25009-13
references.pmid: 12719433
references.citation: Cell Struct Funct. 1998 Feb;23(1):33-42
references.pmid: 9639028
references.citation: Diabetes. 2006 May;55(5):1223-31
references.pmid: 16644676
references.citation: J Biol Chem. 2008 Aug 29;283(35):23542-56
references.pmid: 18566453