Insulin uptake and degradation is a complex and not yet completely understood process involving not only insulin sensitive tissues The most important degradative system is insulin degrading enzyme which is a highly conserved metalloendopeptidase requiring Zn(++) for its proteolytic action, although protein disulfide isomerase and cathepsin D are also involved in insulin metabolism The liver and the kidney are the principal sites for insulin clearance In obese subjects with hyperinsulinemia and high levels of free fatty acids, insulin hepatic clearance is impaired, while the glomerular filtration rate, renal plasma flow and albumin excretion are increased, suggesting a state of renal vasodilatation leading to an abnormally transmitted arterial pressure to the glomerular capillaries through a dilated afferent arteriole Insulin can be cleared also by muscle, adipocytes, gastrointestinal cells, fibroblasts, monocytes and lymphocytes which contain insulin receptors and internalization and regulation mechanism for insulin metabolism

Insulin clearance in obesity

Substrate activation of insulin-degrading enzyme (insulysin). A potential target for drug development.

Structure of Substrate-free Human Insulin-degrading Enzyme (IDE) and Biophysical Analysis of ATP-induced Conformational Switch of IDE

The pancreas is an organ with a central role in nutrient breakdown, nutrient sensing and release of hormones regulating whole body nutrient homeostasis. In diabetes mellitus, the balance is broken—cells can be starving in the midst of plenty. There are indications that the incidence of diabetes type 1 and 2, and possibly pancreatogenic diabetes, is rising globally. Events leading to insulin secretion and action are complex, but there is emerging evidence that intracellular nucleotides and nucleotides are not only important as intracellular energy molecules but also as extracellular signalling molecules in purinergic signalling cascades. This signalling takes place at the level of the pancreas, where the close apposition of various cells—endocrine, exocrine, stromal and immune cells—contributes to the integrated function. Following an introduction to diabetes, the pancreas and purinergic signalling, we will focus on the role of purinergic signalling and its changes associated with diabetes in the pancreas and selected tissues/organ systems affected by hyperglycaemia and other stress molecules of diabetes. Since this is the first review of this kind, a comprehensive historical angle is taken, and common and divergent roles of receptors for nucleotides and nucleosides in different organ systems will be given. This integrated picture will aid our understanding of the challenges of the potential and currently used drugs targeted to specific organ/cells or disorders associated with diabetes.

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Purinergic signalling and diabetes

We studied the ability of ATP to inhibit in vitro the degrading activity of insulin-degrading enzyme. The enzyme was purified from rat skeletal muscle by successive chromatographic steps. The last purification step showed two bands at 110 and 60 kDa in polyacrylamide gel. The enzyme was characterized by its insulin degradation activity, the substrate competition of unlabeled to labeled insulin, the profile of enzyme inhibitors, and the recognition by a specific antibody. One to 5 mM ATP induced a dose-dependent inhibition of insulin degradation (determined by trichloroacetic acid precipitation and insulin antibody binding). Inhibition by 3 mM adenosine 5'-diphosphate, adenosine 5'-monophosphate, guanosine 5'-triphosphate, pyrophosphate, beta-gamma-methyleneadenosine 5'-triphosphate, adenosine 5'-O-(3 thiotriphosphate), and dibutiryl cyclic adenosine 5'-monophosphate was 74%, 4%, 38%, 46%, 65%, 36%, and 0%, respectively, of that produced by 3 mM ATP. Kinetic analysis of ATP inhibition suggested an allosteric effect as the plot of 1/v (insulin degradation) versus ATP concentration was not linear and the Hill coefficient was more than 1 (1.51 and 2.44). The binding constant for allosteric inhibition was KiT = 1.5 x 10(-7) M showing a decrease of enzyme affinity induced by ATP. We conclude that ATP has an inhibitory effect on the insulin degradation activity of the enzyme.

ATP inhibits insulin-degrading enzyme activity.

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.

I - insulin transfer to 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 25 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

María I. Wanderley

Papers

ATP inhibits insulin-degrading enzyme activity.

I - insulin transfer to mitochondria.

II - Insulin processing in mitochondria