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.

I - insulin transfer to mitochondria.

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.

Foetal exposure to Panax ginseng extract reverts the effects of prenatal dexamethasone in the synthesis of testosterone by Leydig cells of the adult rat.

The aim of this study was to examine the effect of maternal exposure to Panax ginseng extract (GE) on the prenatal dexamethasone (DEXA)-induced increase in testosterone production by isolated Leydig cells in adult rats. Pregnant rats were treated with (i) GE (200 mg/kg) or vehicle on days 10-21; (ii) DEXA (100 µg/kg) or vehicle on days 14-21; or (iii) a combination of GE plus DEXA at the same doses and with the same regimen. Testosterone production was induced either by the activator of protein kinase A (dbcAMP) or substrates of steroidogenesis [22(R)-hydroxycholesterol (22(R)-OH-C)] and pregnenolone. The capacity of rat Leydig cells exposed to DEXA to synthesize testosterone induced by dbcAMP, 22(R)-OH-C or pregnenolone was increased in comparison with the control group. Combined exposure to DEXA + GE prevented the effect of DEXA on the responsiveness of Leydig cells to all inductors of testosterone synthesis, whereas GE alone did not modify the response to inductors. No modifications in testosterone production were observed under basal conditions. StAR immunoexpression in Leydig cells was not modified by prenatal exposure to DEXA, GE or DEXA + GE. P450scc and glucocorticoid receptor immunoexpression was higher in offspring exposed to DEXA in comparison with the control group. This increased expression was prevented by combined treatment with DEXA + GE. The present findings demonstrate that GE is capable of reversing the effect of DEXA on testosterone synthesis by rat Leydig cells.

Effect of dexamethasone and testosterone treatment on the regulation of insulin-degrading enzyme and cellular changes in ventral rat prostate after castration.

Insulin-degrading enzyme (IDE) has been shown to enhance the binding of androgen and glucocorticoid receptors to DNA in the nuclear compartment. Glucocorticoids cause hyperglycaemia, peripheral resistance to insulin and compensatory hyperinsulinaemia. The aim of the present study was to investigate the effect of dexamethasone (D), testosterone (T) and dexamethasone plus testosterone (D + T) on the regulation of IDE and on the remodelling of rat ventral prostate after castration (C). Castration led to a marked reduction in prostate weight (PW). Body weight was significantly decreased in the castrated animals treated with dexamethasone, and the relative PW was 2.6-fold (±0.2) higher in the D group, 2.8-fold (±0.3) higher in the T group and 6.6-fold (±0.6) higher in the D + T group in comparison with the castrated rats. Ultrastructural alterations in the ventral prostate in response to androgen deprivation were restored after testosterone and dexamethasone plus testosterone treatments and partially restored with dexamethasone alone. The nuclear IDE protein level indicated a 4.3-fold (±0.4) increase in castrated rats treated with D + T when compared with castration alone. Whole-cell IDE protein levels increased approximately 1.5-fold (±0.1), 1.5-fold (±0.1) and 2.9-fold (±0.2) in the D, T and D + T groups, respectively, when compared with castration alone. In conclusion, the present study reports that dexamethasone-induced hyperinsulinaemic condition plus exogenous testosterone treatment leads to synergistic effects of insulin and testosterone in the prostatic growth and in the amount of IDE in the nucleus and whole epithelial cell.

Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: in vivo and ex vivo studies.

BACKGROUND: The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats. METHODS: Twelve adult male Wistar rats were treated with rosiglitazone (5 mg/kg) administered by gavage for 15 days. Twelve control animals were treated with the vehicle. The ability of rosiglitazone to directly affect the production of testosterone by Leydig cells ex vivo was evaluated using isolated Leydig cells from rosiglitazone-treated rats. Testosterone production was induced either by activators of the cAMP/PKA pathway (hCG and dbcAMP) or substrates of steroidogenesis [22(R)-hydroxy-cholesterol (22(R)-OH-C), which is a substrate for the P450scc enzyme, and pregnenolone, which is the product of the P450scc-catalyzed step]. Testosterone in plasma and in incubation medium was measured by radioimmunoassay. The StAR and P450scc expression was detected by immunocytochemistry. RESULTS: The levels of total circulating testosterone were not altered by rosiglitazone treatment. A decrease in basal or induced testosterone production occurred in the Leydig cells of rosiglitazone-treated rats. The ultrastructural and immunocytochemical analysis of Leydig cells from rosiglitazone-treated rats revealed cells with characteristics of increased activity as well as increased StAR and P450scc expression, which are key proteins in androgen biosynthesis. However, a number of rosiglitazone-treated cells exhibited significant mitochondrial damage. CONCLUSION: The results revealed that the Leydig cells from rosiglitazone-treated rats showed significant reduction in testosterone production under basal, hCG/dbcAMP- or 22 (R)-OH-C/pregnenolone-induced conditions, although increased labeling of StAR and P450scc was detected in these cells by immunocytochemistry. The ultrastructural study suggested that the lower levels of testosterone produced by these cells could be due to mitochondrial damage induced by rosiglitazone.

Green tea polyphenols inhibit testosterone production in rat Leydig cells.

This study investigated the acute effects of green tea extract (GTE) and its polyphenol constituents, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin (EC), on basal and stimulated testosterone production by rat Leydig cells in vitro. Leydig cells purified in a Percoll gradient were incubated for 3 h with GTE, EGCG or EC and the testosterone precursor androstenedione, in the presence or absence of either protein kinase A (PKA) or protein kinase C (PKC) activators. The reversibility of the effect was studied by pretreating cells for 15 min with GTE or EGCG, allowing them to recover for 1 h and challenging them for 2 h with human chorionic gonadotropin (hCG), luteinizing hormone releasing hormone (LHRH), 22(R)-hydroxycholesterol or androstenedione. GTE and EGCG, but not EC, inhibited both basal and kinase-stimulated testosterone production. Under the pretreatment conditions, the inhibitory effect of the higher concentration of GTE/EGCG on hCG/LHRH-stimulated or 22(R)-hydroxycholesterol-induced testosterone production was maintained, whereas androstenedione-supported testosterone production returned to control levels. At the lower concentration of GTE/EGCG, the inhibitory effect of these polyphenols on 22(R)-hydroxycholesterol-supported testosterone production was reversed. The inhibitory effects of GTE may be explained by the action of its principal component, EGCG, and the presence of a gallate group in its structure seems important for its high efficacy in inhibiting testosterone production. The mechanisms underlying the effects of GTE and EGCG involve the inhibition of the PKA/PKC signalling pathways, as well as the inhibition of P450 side-chain cleavage enzyme and 17beta-hydroxysteroid dehydrogenase function.

Androgen- and estrogen-dependent regulation of insulin-degrading enzyme in subcellular fractions of rat prostate and uterus.

Innumerous data support the fact that insulin-degrading enzyme (IDE) is the primary enzymatic mechanism for initiating and controlling cellular insulin degradation. Nevertheless, insulin degradation is unlikely to be the only cellular function of IDE, because it appears that some cellular effects of insulin are mediated by IDE as a regulatory protein. Insulin-degrading enzyme shows a significant correlation with various cellular functions, such as cellular growth and differentiation, and the expression of IDE is developmentally regulated. Besides insulin, other substrates are also degraded by IDE, including various growth-promoting peptides. It has also been shown that IDE enhances the binding of androgen to DNA in the nuclear compartment. It is also known that the androgen hormones have a stimulatory effect on prostate growth, and that estradiol stimulates uterine growth. To establish whether IDE is regulated by a cellular prostate/uterine growth stimulus, the present study assessed whether IDE was modified in quantity and activity during proliferative conditions (castration + testosterone in the male rat, or castration + estradiol or the proestrus phase of the estrous cycle in the female rat) and autolysis (castration or the metestrus phase of the estrous cycle) using cytosolic and nuclear fractions of rat prostate and cytosolic fractions of rat uterus. The activity and amount of IDE decreased in the cytosolic fraction with castration and during metestrus, and increased with testosterone or estradiol treatment and during proestrus. In the nuclear fraction, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although without degradative activity. The data presented here suggest that IDE may participate in prostatic and uterine growth and that the testosterone or estradiol and/or prostate and uterus insulin-like growth factors may be important factors for the expression and regulation of IDE in the prostate and uterus.