1α,25-(OH)2 vitamin D3 prevents insulin resistance and regulates coordinated exocytosis and insulin secretion.

Vitamin D3 is associated with improvements in insulin resistance and glycemia. In this study, we investigated the short-term effect of 1α,25(OH)2 Vitamin D3 (1,25-D3) and cholecalciferol (vitamin D3) on the glycemia and insulin sensitivity of control and dexamethasone-induced insulin-resistance rats. 45Ca2+ influx responses to 1,25-D3 and its role in insulin secretion were investigated in isolated pancreatic islets from control rats. In vivo, 5 d treatment with 1,25-D3 (i.p.) prevented insulin resistance in dexamethasone-treated rats. Treatment with 1,25-D3 improved the activities of hepatic enzymes, serum lipids and calcium concentrations in insulin-resistant rats. 25-D3 (o.g.) does not affect insulin resistance. In pancreatic islets, 1,25-D3 increased insulin secretion and stimulated rapid response 45Ca2+ influx. The stimulatory effect of 1,25-D3 on 45Ca2+ influx was decreased by diazoxide, apamine, thapsigargin, dantrolene, 2-APB, nifedipine, TEA, PKA, PKC, and cytoskeleton inhibitor, while it was increased by glibenclamide and N-ethylmaleimide. The stimulatory effect of 1,25-D3 on 45Ca2+ influx involves the activation of L-type VDCC, K+-ATP, K+-Ca2+, and Kv channels, which augment cytosolic calcium. These ionic changes mobilize calcium from stores and downstream activation of PKC, PKA tethering vesicle traffic and fusion at the plasma membrane for insulin secretion. This is the first study highlighting the unprecedented role of 1,25-D3 (short-term effect) in the regulation of glucose homeostasis and on prevention of insulin resistance. Furthermore, this study shows the intracellular ß-cell signal transduction of 1,25-D3 through the modulation of pivotal ionic channels and proteins exhibiting a coordinated exocytosis of vesicles for insulin secretion.

Crosstalk in the non-classical signal transduction of testosterone and retinol in immature rat testes.

This study aimed to investigate the effects of the interaction between testosterone and retinol on the rapid responses of cultured Sertoli cells obtained from 10-day-old immature rat testes. Non-classical actions of testosterone and retinol were investigated, and the activities of L-type voltage-dependent calcium channels (L-VDCC) and voltage-dependent potassium channels (Kv) were determined by measuring 45Ca2+ influx in whole testis. Additionally, the effects of testosterone and retinol on these channels were studied in primary culture of Sertoli cells using the patch-clamp technique. 45Ca2+ influx was used to observe a dose-response curve on tissues treated with retinol and/or testosterone for 2 min (10-12, 10-9 and 10-6 M and 10-9 and 10-6 M), and a concentration of 10-6 M was selected to investigate the mechanism of action of testosterone and retinol on rapid responses. Participation of the L-VDCC and Kv channels was investigated using nifedipine and tetraethylammonium chloride (TEA) inhibitors, respectively. Both, testosterone and retinol act through non-classical mechanisms, stimulating 45Ca2+ influx in immature rat testes. The response to testosterone was abolished by nifedipine and TEA, whereas the effects of retinol were partially blocked by nifedipine and completely inhibited by TEA. Retinol amplified the testosterone-induced effect on 45Ca2+ influx in the testes, suggesting a crosstalk between rapid responses (calcium influx) and cell repolarization via activation of Kv channels. Whole-cell electrophysiology data demonstrated that testosterone and retinol increased voltage-dependent potassium currents (Kv) in Sertoli cells; inhibition of these responses by TEA confirmed the involvement of TEA-sensitive K+ channels in these effects. Taken together, we demonstrate, for the first time, crosstalk between testosterone and retinol that is mediated by a non-classical mechanism involving the L-VDCC-triggered cell depolarization and activation of repolarization by Kv currents in Sertoli cells. These ionic modulations play a physiological role in Sertoli cells and male fertility via stimulation of secretory activities.

Acute effect of bisphenol A: Signaling pathways on calcium influx in immature rat testes.

We investigated the acute effect of low concentrations of BPA on calcium influx and the mechanism of action of BPA in this rapid response in the rat testis. BPA increased calcium influx at 1 pM and 1 nM at 300 s of incubation, in a similar manner to that of estradiol. At 1 pM, BPA stimulated calcium influx independently of classical estrogen receptors, consistent with a G-protein coupled receptor. This effect also involves the modulation of ionic channels, such as K+, TRPV1 and Cl- channels. Furthermore, BPA is able to modulate calcium from intracellular storages by inhibiting SERCA and activating IP3 receptor/Ca2+ channels at the endoplasmic reticulum and activate kinase proteins, such as PKA and PKC. The rapid responses of BPA on calcium influx could, in turn, trigger a cross talk by MEK and p38MAPK activation and also mediate genomic responses.

Epitestosterone- and testosterone-replacement in immature castrated rats changes main testicular developmental characteristics.

Epitestosterone is the 17α-epimer of testosterone and has been described as an anti-androgen, since it inhibits the effects produced by testosterone and dihydrotestosterone via the nuclear androgen receptor (nAR). However, epitestosterone also displays an effect which is similar to the non-classical effect of testosterone, depolarizing the membrane potential of Sertoli cells and inducing a rapid Ca2+ uptake. This study aimed to investigate the effects of a treatment with epitestosterone on developmental parameters of immature rats. Animals were chemically castrated by using the gonadotropin-releasing hormone (GnRH) antagonist cetrorelix and then received a replacement of 7 days with epitestosterone or testosterone. Replacement with either epitestosterone or testosterone restored the anogenital distance (AGD) and testicular weight which had been reduced by chemical castration. The immunocontent of nAR and the nAR-immunoreactivity were reduced by epitestosterone treatment in the testis of both castrated and non-castrated animals. Furthermore, testosterone was unable of changing the membrane potential of Sertoli cells through its non-classical action in the group of animals castrated and replaced with epitestosterone. In conclusion, in relation to the level of protein expression of nAR epitestosterone acts as an anti-androgen. However, it acts in the same way as testosterone when genital development parameters are evaluated. Moreover, in castrated rats epitestosterone suppressed the non-classical response of testosterone, changing the pattern of testosterone signalling via a membrane mechanism in Sertoli cells.

Non-classical effects of androgens on testes from neonatal rats.

The intratesticular testosterone concentration is high during the early postnatal period although the intracellular androgen receptor expression (iAR) is still absent in Sertoli cells (SCs). This study aimed to evaluate the non-classical effects of testosterone and epitestosterone on calcium uptake and the electrophysiological effects of testosterone (1µM) on SCs from rats on postnatal day (pnd) 3 and 4 with lack of expression of the iAR. In addition, crosstalk on the electrophysiological effects of testosterone and epitestosterone with follicle stimulating hormone (FSH) in SCs from 15-day-old rats was evaluated. The isotope (45)Ca(2+) was utilized to evaluate the effects of testosterone and epitestosterone in calcium uptake. The membrane potential of SCs was recorded using a standard single microelectrode technique. No immunoreaction concerning the iAR was observed in SCs on pnd 3 and 4. At this age, both testosterone and epitestosterone increased the (45)Ca(2+) uptake. Testosterone promoted membrane potential depolarization of SCs on pnd 4. FSH application followed by testosterone and epitestosterone reduced the depolarization of the two hormones. Application of epitestosterone 5 min after FSH resulted in a delay of epitestosterone-promoted depolarization. The cell resistance was also reduced. Thus, in SCs from neonatal Wistar rats, both testosterone and epitestosterone act through a non-classical mechanism stimulating calcium uptake in whole testes, and testosterone produces a depolarizing effect on SC membranes. Testosterone and epitestosterone stimulates non-classical actions via a membrane mechanism, which is independent of iAR. FSH and testosterone/epitestosterone affect each other's electrophysiological responses suggesting crosstalk between the intracellular signaling pathways.

Non-classic androgen actions in Sertoli cell membrane in whole seminiferous tubules: effects of nandrolone decanoate and catechin.

Studies show a mechanism of action of testosterone, nandrolone and catechin as agonists of the membrane androgen receptor. The aim of this work is to investigate the non-classical effect of androgens and catechin in Sertoli cells from immature rats. The membrane potential of Sertoli cells in whole seminiferous tubules was recorded using a standard single microelectrode technique. It was performed a topical application of testosterone (1 µM), nandrolone (0.1, 0.5 and 1 µM) and the flavonoid catechin (0.1, 0.5 and 1 µM) alone and also after infusion with flutamide (1 µM), diazoxide (100 µM) or U73122 (1 µM). The immature testes were incubated for 5 min in KRb with (45)Ca(2+), with or without nandrolone (1 µM). The results were given as mean±SEM. The data were analyzed using ANOVA for repeated measures with Bonferroni post-test. Testosterone produces a depolarization in the membrane potential at 120 s after application. Catechin (1 µM) and nandrolone (1 µM) have shown a similar response to testosterone: depolarization at 120 s after the application. The same response of catechin and nandrolone was observed at different doses. The effects of testosterone, catechin and nandrolone were not affected after perfusion with flutamide. Perfusion with diazoxide and U73122 nullified the effect of nandrolone (1 µM) and catechin (1 µM). Nandrolone and testosterone increased (45)Ca(2+) uptake with or without flutamide within 5min. These results indicate that nandrolone and catechin act through a receptor on the plasmatic membrane, as well as testosterone, showing a non-classical pathway in Sertoli cells from immature rat testes.