Skip to main content
SpringerLink
Log in

I - insulin transfer to mitochondria

  • Published:
Journal of Bioenergetics and Biomembranes Aims and scope Submit manuscript

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 125I-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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

IDE :

Insulin-degrading enzyme

M :

mitoplasts

OM :

outer membrane

IMS :

inter-membrane space

IM :

inner membrane

Mx :

matrix

OM + IMS :

outer membrane + inter-membrane space

DNP :

2,4-dinitrophenol

NEM :

N-ethylmaleimide

TIM23 :

inner mitochondrial transporter protein

References

  • Allard C, de Lamirande G, Cantero A (1952) Mitochondrial population in mammalian cells (liver). Cancer Res 12:580–83

    CAS  Google Scholar 

  • Aprille JR (1988) Regulation of the mitochondrial adenine nucleotide pool size in liver: mechanism and metabolic role. FASEB J 2:2547–2556

    CAS  Google Scholar 

  • Authier F, Bergeron JJM, Ou WJ, Rachubinski RA, Posner BI, Walton RA (1995) Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase. Proc Natl Acad Sci U S A 92:3859–3863

    Article  CAS  Google Scholar 

  • Bennett RG, Hamel FG, Duckworth WC (1997) Characterization of the insulin inhibition of the peptidolytic activities of the insulin degrading enzyme-proteasome complex. Diabetes 46:197–203

    Article  CAS  Google Scholar 

  • Camberos MC, Cresto JC (1982) Caracterización de sueros anti-insulina para la determinación de insulina y proinsulina en pacientes. Acta Bioquim Clin Latinoam 16:453–457

    CAS  Google Scholar 

  • Camberos MC, Cresto JC (2007) Insulin-degrading enzyme hydrolyzes ATP. Exp Biol Med 232:281–292

    CAS  Google Scholar 

  • Camberos MC, Perez AA, Udrisar DP, Wanderley MI, Cresto JC (2001) ATP inhibits insulin-degrading enzyme activity. Exp Biol Med 226:334–341

    CAS  Google Scholar 

  • Caruso M, Maitan MA, Bifulco G, Miele C, Vigliotta G, Oriente F, Formisano P, Beguinot F (2001) Activation and mitochondrial translocation of protein kinase Cδ are necessary for insulin stimulation of pyruvate dehydrogenase complex activity in muscle and liver cells. J Biol Chem 276:45088–45097

    Article  CAS  Google Scholar 

  • César Vieira JSB, Saraiva KLA, Barbosa MCL, Porto RCC, Cresto JC, Peixoto CA, Wanderley MI, Udrisar DP (2011) Effect of dexamethasone and testosterone treatment on the regulation of insulin-degrading enzyme and cellular changes in ventral rat prostate after castration. Int J Exp Path 92:272–280

    Article  Google Scholar 

  • Cresto JC, Udrisar DP, Camberos MC, Basabe JC, Gomez Acuña P, de Majo SF (1981) Preparation of biologically active mono-125I-insulin of high specific activity. Acta Physiol Lat 31:13–24

    CAS  Google Scholar 

  • Duckworth WC, Bennett RG, Hamel FG (1998) Insulin degradation: progress and potential. Endocr Rev 19:608–624

    CAS  Google Scholar 

  • Ezaki O, Kono T (1982) Effects of temperature on basal and insulin-stimulated glucose transport activities in fat cells - Further support for the translocation hypothesis on insulin action. J Biol Chem 257:14306–14310

    CAS  Google Scholar 

  • Glick BS, Wachter C, Reid GA, Schatz G (1993) Import of cytochrome b 2 to the mitochondrial intermembrane space: The tightly folded hame-binding domain makes import dependent upon matrix ATP. Protein Sci 2:1901–1917

    Article  CAS  Google Scholar 

  • Guyton AC, Hall JE: Tratado de Fisiología Humana (1997) Editor McGraw-Hill, Interamericana, Mexico Chapter 70, 961-967

  • Haigis MC, Mostoslavsky R, Haigis KM, Fahie K, Christodoulou DC, Murphy AJ, Valenzuela DM, Yancopoulos GD, Karow M, Blander G, Wolberger C, Prolla TA, Weindruch R, Alt FW, Guarente L (2006) SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells. Cell 126:941–954

    Article  CAS  Google Scholar 

  • Hamel FG, Mahoney MJ, Duckworth WC (1991) Degradation of intraendosomal insulin by insulin-degrading enzyme without acidification. Diabetes 40:436–443

    Article  CAS  Google Scholar 

  • Hamel FG, Bennet RG, Upward JL, Duckworth WC (2001) Insulin inhibits peroxisomal fatty acid oxidation in isolated rat hepatoxytes. Endocrinology 142:2702–2706

    CAS  Google Scholar 

  • Ho YS, McKay G (1999) Pseudo-second order model for sorption processes. Process Biochem 34:451–465

    Article  CAS  Google Scholar 

  • Hüttemann M, Icksoo L, Samavati L, Hong Y, Doan JW (2007) Regulation of mitochondrial oxidative phosphorylation through cell signaling. Biochim Biophys Acta 1773:1701–1720

    Article  Google Scholar 

  • Im H, Manolopoulou M, Malito E, Shen Y, Zhao J, Neant-Fery M, Sun MS, Sisodia SS, Leissring MA, Tang WJ (2007) Structure of substrate-free human insulin-degrading enzyme (IDE) biophysical analysis of ATP-induced conformational switch of IDE. J Biol Chem 282:25453–25463

    Article  CAS  Google Scholar 

  • Lachmanovich E, Svartsman DE, Malka Y, Botvin C, Henis YI, Weiss AM (2003) Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies. J Microsc 212:122–131

    Article  CAS  Google Scholar 

  • Leal MC, Magnani N, Villordo S, Buslje CM, Evelson P, Castaño EM, Morelli L (2013) Transcriptional regulation of insulin-degrading enzyme modulates mitochondrial amyloid β (Aβ) peptide catabolism and functionality. J Biol Chem 288:12920–12931

    Article  CAS  Google Scholar 

  • Leissring MA, Farris W, Wu X, Christodoulou DC, Haigis MC, Guarente L, Selkoe DJ (2004) Alternative translation initiation generates a novel isoform of insulin-degrading enzyme targeted to mitochondria. Biochem J 383:439–446

    Article  CAS  Google Scholar 

  • Mokranjac D, Neupert W (2005) Protein import into mitochondria. Biochem Soc Trans 33:1019–1023

    Article  CAS  Google Scholar 

  • Mylotte LA, Duffy AM, Murphy M, O’Brien T, Samali A, Barry F, Szegezdi E (2008) Metabolic flexibility permits mesenchymal stem cell survival in an ischemic environment. Stem Cells 26:1325–1336

    Article  CAS  Google Scholar 

  • Rickwood D, Wilson MT, Darley-Usmar VM (1987) Isolation and characteristics of intact mitochondria. In: Mitochondria, a practical approach. Editor: Darley-Usmar VM, Rickwood D, Wilson MT, IRL Press Limited. Chapter 1, pp. 1-16.

  • Rosenzweig JL, Havrankova J, Lesniak MA, Brownstein M, Roth J (1980) Insulin is ubiquitous in extra-pancreatic tissues of rats and humans. Proc Natl Acad Sci U S A 77:572–576

    Article  CAS  Google Scholar 

  • Salvi M, Brunati AM, Toninello A (2005) Tyrosine phosphorylation in mitochondria: a new frontier in mitochondrial signaling. Free Radic Biol Med 38:1267–1277

    Article  CAS  Google Scholar 

  • Schwartz MP, Huang S, Matouschek A (1999) The structure of precursor proteins during import into mitochondria. J Biol Chem 274:12759–12764

    Article  CAS  Google Scholar 

  • Udrisar DP, Wanderley MI, Porto RCC, Cardoso CLP, Barbosa CL, Camberos MC, Cresto JC (2005) Androgen and androgen-dependent regulation of insulin-degrading enzyme in sub-cellular fractions of rat prostate and uterus. Exp Biol Med 230:479–487

    CAS  Google Scholar 

  • Van der Laan M, Rissier M, Rehling P (2006) Mitochondrial pre-protein translocases as dynamic molecular machines. FEMS Yeast Res 6:849–861

    Article  Google Scholar 

  • Wymann M, Bulgarelli-keva G, Zvelebil MJ, Pirola L, Vanhaesebroeck B, Waterfield D, Panayotou G (1996) Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cel Biol 16:1722–1733

    CAS  Google Scholar 

  • Yonezawa K, Yokono K, Shii K, Hari J, Yaso S, Amano K, Sakamoto T, Kawase Y, Akiyama H, Nagata M, Baba S (1988) Insulin-degrading enzyme is capable of degrading receptor-bound insulin. Biochem Biophys Res Commun 150:605–614

    Article  CAS  Google Scholar 

Download references

Acknowledgments

All authors participated in the design and interpretation of the studies, as well as in the analysis of the data and review of the work. The authors would like to thank Drs. J.J. Anderson and R. Gabach for their constant significant contribution during the development of this work and Andrea C. Cruz for her skillful help in the English revision.

Author information

Authors and Affiliations

  1. Endocrinology Research Center (CEDIE-CONICET) Children Hospital “R. Gutiérrez”, Gallo 1330, 1425, Ciudad de Buenos Aires, Argentina

    María del Carmen Camberos & Juan C. Cresto

  2. Pathology Division, Children Hospital “P. Elizalde”, Buenos Aires, Argentina

    Gabriel Cao

  3. Endocrinology and Metabolism Laboratory, Physiology and Pharmacology Department, University of Pernambuco, Recife, Pernambuco, Brazil

    María I. Wanderley & Daniel P. Udrisar

Authors
  1. María del Carmen Camberos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabriel Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. María I. Wanderley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel P. Udrisar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan C. Cresto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juan C. Cresto.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

Electron microscopy of mitochondria. Mitochondria were isolated as described in Material and Methods. Contamination after mitochondrial isolation was: LDH (mUI/mg protein, showed as % citosolic concentration) 11.48%; Catalase (mU/mg protein): 7.82%. It is showed a group of mitochondria with conserved double membrane over a background of cisternal membranes. (TEM: 25000x, original magnification). Inset. An isolated mitochondrion is showed with preserved ultra-structure (TEM: 50000x, original magnification). (DOC 1921 kb)

ESM 2

Control of insulin degradation at 25ºC. ■ - Insulin control; □ - IDE+insulin. Insulin was incubated alone or with IDE during 300 sec at 25ºC and subject to chromatography in Sephadex G50 superfine. There was not degraded insulin. (DOC 26 kb)

ESM 3

Second order reaction -Concentration dependence of reactants. [Insulin-IDE] and [Mitoplasts] (n: 2). In the conditions described in Material and Methods, we added 125I-insulin at two concentrations: (■) - 6,67x10-10 M and (▲) - 6,67x10-11 M. It is showed the concentration dependence to one of the reactants [IDE-insulin] as described for a second order reaction. The slope has been calculated as described by Ho and McKay (17). There was a concentration dependence on [IDE-insulin] because the mitoplasts concentration was unchanged. (DOC 24 kb)

ESM 4

Insulin accumulation in outer membrane+intermembrane space (OM+IMS) and mitoplasts (M). A (OM+IMS) (n: 3): Control (◆), IDE (◊), Control+ Apyrase (●), IDE+Apyrase (○). B (M) (n: 3): Control (◆), IDE (◊), Control+Apyrase (●), IDE+Apyrase (○). C (OM+IMS) (n: 3): Control (◆), IDE (◊), Succinate-ADP+IDE (■). D (M) (n: 3): Control (◆), IDE (◊), Succinate-ADP+IDE (■). IDE did not induce insulin accumulation in OM+IMS, but IDE incremented the insulin in M. This increment of insulin in mitoplasts was decreased by apyrase, but it was not significant. Succinate-ADP did not modify the increment of insulin produced by IDE. (DOC 31 kb)

ESM 5

IDE and insulin immunoblots, and immunoblots autoradiography (n: 2). C: control without cross-link. The 119 kDa band was recognized by IDE antibody. S.5A - IDE immunoblot: Mitoplasts, OM+IMS and IDE addition are described in the figure (+ or -). There are two bands at 150 and 136 kDa, which can be observed in all immunoblot. S.5A - Insulin immunoblot: Mitoplasts, OM+IMS and IDE addition are described in the figure (+ or -). Insulin antibody recognizes the same bands of 150 and 136 kDa, showing the coincidence between insulin and IDE antibodies. S.5B - Autoradiography of immunoblots. The immunoblot showed the same differences between OM+ IMS and M observed in gel autoradiography (Fig. 6.1A). In mitoplasts the IDE addition increased the radioactive spots of 136 kDa and 180 kDa. Mitoplasts densitometry presented statistic differences after IDE addition (p <0.001 to p <0.0001). OM+IMS did not show statistic differences. The immunoblot seem to be run in short time, but autoradiography demonstrates that the run was normal and the transferring line can be observed at bottom. Both antibodies stop antigen recognition at 56 kDa. (PDF 69 kb)

ESM 6

Endogenous rat insulin was assayed by RIA after acid-alcohol extraction [Grodsky et al, J Clin Invest, 1960]. Two mM NEM was used to decrease insulin degradation. The results were adjusted to 100% recovery. Insulin concentration was expressed as insulin ng/g wet tissue [Allard et al, Cancer Res, 1952] to compare mitochondria and liver. Rat insulin was assayed by RIA with rat standard and insulin antibody which recognize rat insulin. The coefficient of variation (CV) was, Exp. 1 (n:6): 0.16; Exp. 2 (n: 6): 0.24. Liver. The first column showed direct results without correction for recovery and the second column was adjusted to 100% recovery. Liver insulin extraction was expressed in ng/grams of wet weight. Insulin in liver (without correction, 1st column); no NEM: 3.83±0.66 (n: 5); NEM: 6.30±0.69 (n: 6) *p <0.02. Insulin in liver (corrected, 2nd column); no NEM: 14.82±0.97 (n: 5); NEM: 19.83±1.89 (n: 7), **p <0.04. Liver mitochondria. As in total liver the results were expressed: ng/g liver wet weight, with 100% correction. No NEM: 1.37±0.49 (n: 5); NEM: 3.50±0.97 (n: 6), *p <0.05. (Martucci LC: Thesis for Master´s Degree in Biology. “Estudio de la distribución tisular de insulina endógena en la rata. Su concentración en mitocondrias hepáticas”, Universidad de Belgrano, Buenos Aires, 2011. (Unpublished results, reproduced with permission). (DOC 39 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

del Carmen Camberos, M., Cao, G., Wanderley, M.I. et al. I - insulin transfer to mitochondria. J Bioenerg Biomembr 46, 357–370 (2014). https://doi.org/10.1007/s10863-014-9563-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10863-014-9563-y

Keywords

Search

Navigation