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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.

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.