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II - Insulin processing in mitochondria

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Abstract

Our objective was to know how insulin is processing in mitochondria; if IDE is the only participant in mitochondrial insulin degradation and the role of insulin degradation on IDE accumulation in mitoplasts. Mitochondria and its fractions were isolated as described by Greenwalt. IDE was purified and detected in immunoblot with specific antibodies. High insulin degradation was obtained through addition to rat’s diet of 25 g/rat of apple and 10 g/rat of hard-boiled eggs, 3 days a week. Mitochondrial insulin degradation was assayed with 5 % TCA, insulin antibody or Sephadex G50 chromatography. Degradation was also assayed 60 min at 37 °C in mitochondrial fractions (IMS and Mx) with diet or not and without IDE. Degradation in fractions precipitated with ammonium sulfates (60–80 %) were studied after mitochondrial insulin incubation (1 ng. insulin during 15 min, at 30 °C) or with addition of 2.5 mM ATP. Supplementary diet increased insulin degradation. High insulin did not increase mitoplasts accumulation and did not decrease mitochondrial degradation. High insulin and inhibition of degradation evidence insulin competition for a putative transport system. Mitochondrial incubation with insulin increased IDE in matrix as observed in immunoblot. ATP decreased degradation in Mx and increased it in IMS. Chromatography of IMS demonstrated an ATP-dependent protease that degraded insulin, similar to described by Sitte et al. Mitochondria participate in insulin degradation and the diet increased it. High insulin did not accomplish mitochondrial decrease of degradation or its accumulation in mitoplasts. Mitochondrial incubation with insulin increased IDE in matrix. ATP suggested being a regulator of mitochondrial insulin degradation.

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Abbreviations

IDE:

Insulin-degrading enzyme

OM:

Outer membrane

IMS:

Inter-membrane space

M:

Mitoplasts

IM:

Inner membrane

Mx:

Matrix

NEM:

N-ethylmaleimide

TCA:

Trichloroacetic acid

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Acknowledgments

We thank Dr. R. Gabach for his advice. We would also like to thank A.C. Cruz and L.Karabatas for proofreading the paper and the revised form of the manuscript

Authors’ contributions

All of the authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript. MCC and JCC conducted the experiments. JCC wrote the manuscript. AP, GP, LCM performed the insulin degradation studies. MIW and DPU participated in the antibody production and paper discussion.

Author information

Authors and Affiliations

  1. Endocrinology Research Center “Dr. Cesar Bergada” (CEDIE-CONICET), Endocrinology Division, Children Hospital. R. Gutierrez, Gallo 1330 (C1425), Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina

    María del Carmen Camberos, Gisel A. Passicot, Lucía C. Martucci & Juan C. Cresto

  2. Department Genetic and Evolution, School of Natural and Exact Sciences, University of Buenos Aires (UBA), Buenos Aires, Argentina

    Adriana A. Pérez

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

    María I. Wanderley & Daniel P. Udrisar

Authors
  1. María del Carmen Camberos
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  2. Adriana A. Pérez
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  3. Gisel A. Passicot
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  4. Lucía C. Martucci
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  5. María I. Wanderley
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  6. Daniel P. Udrisar
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  7. Juan C. Cresto
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Corresponding author

Correspondence to María del Carmen Camberos.

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Financing

This work has been supported by means of subsidies granted by Laboratorios Beta S.A. and Fundación Alberto J. Roemmers.

Competing interests

All authors state that they have not been paid for the work and they have not any economic interest on the present work.

Additional information

María del Carmen Camberos (PC: C1425) Adriana Pérez (PC: C1428), Gisel Passicot (PC: C1425), Lucía C. Martucci (PC: C1425), María I. Wanderley (PC: 50670-901), Daniel P. Udrisar (PC: 50670-901).

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Fig. S1

A: Bacitracin competition with endogenous IDE (no enzyme addition). Incubation: 60 s at 30 °C with 125I-insulin ~50000 cpm. Two mg Bacitracin was added to ~1 mg/mitochondria without pre-incubation, and mitochondrial insulin distribution was determined with chromatography in Sephadex G50 superfine. Radioactive areas are expressed as % values of total area. Peak 1: aggregated insulin; Peak 2: free insulin; Peak 3: insulin degraded. ♦ Control chromatography. Peak 1: 3 %, Peak 2: 27 %, Peak 3: 70 %. ◊ Bacitracin chromatography. Peak 1: 3 %, Peak 2: 54 %, Peak 3: 43 %. Insulin degradation was inhibited 27 % by Bacitracin. B: Mitochondrial inhibition of insulin degradation with increased concentrations of N-ethylmaleimide. Incubation: 10000 ng/insulin + ~50000 cpm 125I-insulin was incubated during 60 s with ~1 mg/mitochondria and 0, 0.25, 0.5, 1 mM NEM in Control and IDE. Insulin degradation was assayed through chromatography in Sephadex G50 superfine in mitochondrial incubation buffer. Radioactive areas are expressed as % values of total area. Peak 1: aggregated insulin; Peak 2: free insulin; Peak 3: insulin degraded. Each peak is expressed as % of total radioactivity. NEM at 1 mM did not impede insulin degradation. (PDF 415 kb)

Fig. S2

Immunoblot of mitochondrial fractions. Mitochondrial fractions were isolated as described in Material and Methods. The electrophoresis in SDS-PAGE of each fraction (45 μg/proteins by line) was transferred to nitrocellulose membrane and analyzed with the specific carboxy-terminal IDE antibody p15. Left: molecular weight of immunodetected IDE bands. C: cytosol, OM: outer membrane, IMS: inter-membrane space, IM: inner membrane, Mx: matrix. (PDF 127 kb)

Fig. S3

Mitochondrial and nuclear accumulation of IDE in castrated rats after treatment with testosterone and dexametasone. The method has been described previously (Int. J. Exp. Path. 92: 272–280, 2011). Briefly, castrated rats were treated with testosterone, dexamethasone or both. Rats were euthanized 1 day after the last injection (6 days after castration). The ventral prostate was immediately removed and prepared for ultra-structural and immunocytochemical studies. As observed, hormonal treatment increased IDE in mitochondria and nucleus (Viera et al unpublished results, reproduced with permission). (PDF 32 kb)

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Camberos, M.d.C., Pérez, A.A., Passicot, G.A. et al. II - Insulin processing in mitochondria. J Bioenerg Biomembr 48, 469–482 (2016). https://doi.org/10.1007/s10863-016-9682-8

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