II - Insulin processing in mitochondria
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.
KeywordsIDE Insulin degradation Supplementary diet Chromatography studies Mitochondrial fractions ATP-dependent protease
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
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.
Compliance with ethical standards
This work has been supported by means of subsidies granted by Laboratorios Beta S.A. and Fundación Alberto J. Roemmers.
All authors state that they have not been paid for the work and they have not any economic interest on the present work.
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