title: I - insulin transfer to mitochondria.
abstract: The aim of this study was to determine if insulin is transferred to mitoplasts by insulin-degrading enzyme (IDE).Hepatic mitochondria were isolated and controlled by electron microscopy. IDE was obtained from rats muscle by successive chromatography steps. Insulin accumulation in mitoplasts and outer membrane + intermembrane space (OM + IMS) was studied with (125)I-insulin. Mitochondrial insulin accumulation and degradation was assayed with Sephadex G50 chromatography, insulin antibody and 5 % TCA. Mitoplasts and OM + IMS were isolated with digitonin. Insulin accumulation was studied at 25 °C at different times, without or with IDE, Bacitracin, 2,4-dinitrophenol, apyrase or sodium succinate + adenosine diphosphate. Insulin accumulation in mitoplasts and OM + IMS after mitochondrial cross-linking was studied with electrophoresis in SDS-PAGE, immunoblots of IDE, insulin or TIM23 (inner mitochondrial transporter) and autoradiography.The studies showed that addition of IDE increased insulin transfer from OM + IMS to mitoplasts, and the insulin accumulation in mitoplast was IDE dependent. Bacitracin and 2,4-dinitrophenol decreased this transfer. The [Insulin-IDE] complex and [Mitoplasts] was studied as a bimolecular reaction following a second order reaction. The constant "k" (liter.mol⁻¹ s⁻¹) showed that IDE increased and Bacitracin or 2,4-dinitrophenol decreased the velocity of insulin transfer. SDS-PAGE and immunoblots studies showed bands and radioactivity coincident with IDE, insulin and TIM23. Non degraded insulin was demonstrated in immunoblot after IDE immunoprecipitation from mitoplasts. Confocal studies showed mitochondrial colocalization of IDE and insulin.The results showed that insulin at 25 °C were transferred from OM + IMS to mitoplasts by IDE or that the enzyme facilitates this transfer, and they reach the matrix together.
authors.affiliation: Endocrinology Research Center (CEDIE-CONICET) Children Hospital "R. Gutiérrez", Gallo 1330, 1425, Ciudad de Buenos Aires, Argentina.
authors.forename: María
authors.initials: M
authors.lastname: Del Carmen Camberos
authors.forename: Gabriel
authors.initials: G
authors.lastname: Cao
authors.forename: María I
authors.initials: MI
authors.lastname: Wanderley
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Udrisar
authors.forename: Juan C
authors.initials: JC
authors.lastname: Cresto
mesh_terms: D000818:Animals; D007328:Insulin / Q000378:metabolism*; D007339:Insulysin / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D008930:Mitochondria, Liver / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D007328:Insulin; D007339:Insulysin
references.citation: J Biol Chem. 2007 Aug 31;282(35):25453-63
references.pmid: 17613531
references.citation: J Biol Chem. 2001 Nov 30;276(48):45088-97
references.pmid: 11577086
references.citation: Mol Cell Biol. 1996 Apr;16(4):1722-33
references.pmid: 8657148
references.citation: FEMS Yeast Res. 2006 Sep;6(6):849-61
references.pmid: 16911507
references.citation: J Biol Chem. 1999 Apr 30;274(18):12759-64
references.pmid: 10212260
references.citation: FASEB J. 1988 Jul;2(10):2547-56
references.pmid: 3290024
references.citation: J Biol Chem. 2013 May 3;288(18):12920-31
references.pmid: 23525105
references.citation: Biochim Biophys Acta. 2007 Dec;1773(12):1701-20
references.pmid: 18240421
references.citation: Biochem J. 2004 Nov 1;383(Pt. 3):439-46
references.pmid: 15285718
references.citation: Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3859-63
references.pmid: 7731996
references.citation: Diabetes. 1997 Feb;46(2):197-203
references.pmid: 9000694
references.citation: Int J Exp Pathol. 2011 Aug;92(4):272-80
references.pmid: 21507087
references.citation: J Biol Chem. 1982 Dec 10;257(23):14306-10
references.pmid: 6754726
references.citation: Stem Cells. 2008 May;26(5):1325-36
references.pmid: 18308942
references.citation: J Microsc. 2003 Nov;212(Pt 2):122-31
references.pmid: 14629561
references.citation: Cell. 2006 Sep 8;126(5):941-54
references.pmid: 16959573
references.citation: Free Radic Biol Med. 2005 May 15;38(10):1267-77
references.pmid: 15855046
references.citation: Protein Sci. 1993 Nov;2(11):1901-17
references.pmid: 8268801
references.citation: Exp Biol Med (Maywood). 2001 Apr;226(4):334-41
references.pmid: 11368426
references.citation: Exp Biol Med (Maywood). 2007 Feb;232(2):281-92
references.pmid: 17259336
references.citation: Biochem Soc Trans. 2005 Nov;33(Pt 5):1019-23
references.pmid: 16246036
references.citation: Endocr Rev. 1998 Oct;19(5):608-24
references.pmid: 9793760
references.citation: Cancer Res. 1952 Aug;12(8):580-3
references.pmid: 14945049
references.citation: Biochem Biophys Res Commun. 1988 Jan 29;150(2):605-14
references.pmid: 3277630
references.citation: Proc Natl Acad Sci U S A. 1980 Jan;77(1):572-6
references.pmid: 6987656
references.citation: Endocrinology. 2001 Jun;142(6):2702-6
references.pmid: 11356722
references.citation: Acta Physiol Lat Am. 1981;31(1):13-24
references.pmid: 6765006
references.citation: Exp Biol Med (Maywood). 2005 Jul;230(7):479-86
references.pmid: 15985623
references.citation: Diabetes. 1991 Apr;40(4):436-43
references.pmid: 2010043