Some ultrastructural effects of testosterone and insulin on the ventral prostate of rats in organ culture

Summary The fine structure of the secretory epithelial cells of rat's ventral prostate has been studied following organ culture. Culturing with either testosterone or insulin alone, and with the two hormones combined, were carried out to investigate how insulin modifies the action of testosterone on the maintenance of cellular integrity. After 4 days in hormone-free culture, the secretory epithelial cells showed signs of cellular atrophy and regression, involving loss of the apical microvilli, absence of the apical secretory vacuoles, atrophy of the Golgi apparatus, decrease in rough endoplasmic reticulum and the appearance of autophagic vacuoles. The presence in the medium of either testosterone or insulin alone, or combined, prevented cellular atrophy and regression. The best maintenance of cellular integrity was obtained in a culture containing both hormones. The effects of insulin was approximately equivalent to those of testosterone in the maintenance of cellular integrity.     

Current state of practice regarding testosterone supplementation therapy in men with prostate cancer

Hypogonadal men are characterized by low serum testosterone and symptoms of low energy, decreased libido, and muscle mass as well as impaired concentration and sexual functioning. Men with prostate cancer (PCa) currently on active surveillance or post-therapy, have traditionally been excluded from management paradigms given the decade-old concern that testosterone caused PCa growth. However, there appears to be little or no relationship between serum testosterone concentration and PCa. Androgen action in the prostate has long been known to be affected by the kinetics of receptor saturation and, as such, testosterone beyond a certain baseline is unable to stimulate prostatic growth due to complete intra-prostatic androgen receptor binding. Given this physiologic concept, many clinical investigators have begun to promote testosterone supplementation therapy (TST) as safe in men with PCa. This review examines the basics of testosterone physiology and summarizes the most recent findings on the use of TST in men with PCa on active surveillance and following treatment with external beam radiotherapy, brachytherapy and radical prostatectomy.     

DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation

The majority of human prostate cancer cell lines, including the two "classical" cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299-315), but not the variant N- or C-terminal region (amino acids 1-20 and 900-919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21(CIP1) protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.     

Relative digit lengths and testosterone levels in Guinea baboons

A growing body of literature suggests that the ratio of the lengths of the second to fourth digits (2D:4D) on human hands is sexually dimorphic and associated with prenatal exposure to gonadal hormones, circulating serum testosterone, and a number of psychological and behavioral measures. Little research has investigated digit ratios in nonhuman species. In the present study, we investigated sex differences in digit ratios and their possible association with serum testosterone in a captive group of Guinea baboons (Papio papio). Contrary to the sex difference typically reported in humans, male baboons exhibited a substantially larger 2D:4D than did female baboons. Consistent with the human data, however, lower 2D:4D was associated with higher serum testosterone among the males. The present findings suggest that the relationship between digit ratios and male gonadal hormones may be phylogenetically well-conserved, although they also suggest possible species differences in the causal relationships between developmental mechanisms and sex-differentiated digit length patterns.     

睾酮与肠道菌群关系的研究进展

近年来,随着分子生物学、基因组学、生物信息学分析技术和高通量测序技术的发展,人们对肠道菌群有了新的认识。大量证据表明肠道菌群的变化或不稳定以及多样性改变是一系列肠道功能紊乱及代谢和免疫疾病的结果。目前发现睾酮与代谢相关性疾病相关,睾酮的合成和代谢紊乱将通过相应的代谢通路导致一系列的代谢相关性疾病。随着研究的深入,人们发现睾酮与肠道菌群之间存在着密切的关系,通过相互影响和相互作用,导致机体发生病理生理变化。本文将重点探讨肠道菌群与睾酮的关系,并阐述这种关系在高血压等代谢相关性疾病（如糖尿病、胰岛素抵抗和肥胖）中的作用机制,为疾病的研究和探索提供新的视角和思路。

     

Transforming growth factors and the regulation of cell proliferation

The number of different growth regulatory molecules which have been isolated and characterized is continuing to increase. As more information is obtained, it has become apparent that the cooperative actions of many factors with distinct activities is necessary for appropriate proliferative responses. An interplay of both growth stimulatory and growth inhibitory factors is essential for normal growth. Of crucial importance, therefore, is the appropriate regulation of growth factors. Unregulated expression, synthesis, posttranslational processing or activation of either positive or negative growth signals may contribute to neoplastic transformation (Fig. 3). Altered responses to normally positive or negative signals by transformed cells have been demonstrated by several investigators [64, 79, 84]. While altered growth factor responses in transformed cells are well documented, the mechanisms responsible for the loss of growth control are poorly understood and are likely to be both complex and numerous. Continued efforts to dissect and comprehend fully growth factor action on normal cells will be necessary before an understanding of neoplastic transformation can be achieved.     

Salivary testosterone does not predict mental rotation performance in men or women

Multiple studies report relationships between circulating androgens and performance on sexually differentiated spatial cognitive tasks in human adults, yet other studies find no such relationships. Relatively small sample sizes are a likely source of some of these discrepancies. The present study thus tests for activational effects of testosterone (T) using a within-participants design by examining relationships between diurnal fluctuations in salivary T and performance on a male-biased spatial cognitive task (Mental Rotation Task) in the largest sample yet collected: 160 women and 177 men. T concentrations were unrelated to within-sex variation in mental rotation performance in both sexes. Further, between-session learning-related changes in performance were unrelated to T levels, and circadian changes in T were unrelated to changes in spatial performance in either sex. These results suggest that circulating T does not contribute substantially to sex differences in spatial ability in young men and women. By elimination, the contribution of androgens to sex differences in human performance on these tasks may be limited to earlier, organizational periods.     

Time and amplitude regulation of pulsatile insulin secretion by triggering and amplifying pathways in mouse islets

Glucose-induced insulin secretion is pulsatile. We investigated how the triggering pathway (rise in β-cell [Ca²⁺]_i) and amplifying pathway (greater Ca²⁺ efficacy on exocytosis) influence this pulsatility. Repetitive [Ca²⁺]_i pulses were imposed by high K⁺+ diazoxide in single mouse islets. Insulin secretion (measured simultaneously) tightly followed [Ca²⁺]_i changes. Lengthening [Ca²⁺]_i pulses increased the duration but not the amplitude of insulin pulses. Increasing glucose (5–20 mmol/l) augmented the amplitude of insulin pulses without changing that of [Ca²⁺]_i pulses. Larger [Ca²⁺]_i pulses augmented the amplitude of insulin pulses at high, but not low glucose. In conclusion, the amplification pathway ensures amplitude modulation of insulin pulses whose time modulation is achieved by the triggering pathway.     

Doxazosin reduces cell proliferation and increases collagen fibers in rat prostatic lobes

We investigated the effects of doxazosin (Dox), an alpha-adrenoceptor antagonist used clinically for the treatment of benign prostatic hyperplasia (BPH), on the rat prostatic complex by assessing structural parameters, collagen fiber content, cell proliferation, and apoptosis. Adult Wistar rats were treated with Dox (25 mg/kg per day), and the ventral (VP), dorsolateral, and anterior prostate (AP) regions of the prostate complex were excised at 3, 7, and 30 days after treatment. At 24 h before being killed, the rats were injected once with 5-bromodeoxyuridine (BrdU; thymidine analog) to label mitotically active cells. The prostates were weighed and processed for histochemistry, morphometry-stereology, immunohistochemistry for BrdU, Western blotting for proliferating cell nuclear antigen (PCNA), and the TUNEL reaction for apoptosis. Dox-treated prostate lobes at day 3 presented increased weight, an enlarged ductal lumen, low cubical epithelial cells, reduced epithelial folds, and stretched smooth muscle cells. However, at day 30, the prostates exhibited a weight reduction of ∼20% and an increased area of collagen and reticular fibers in the stromal space. Dox also reduced epithelial cell proliferation and increased apoptosis in the three prostatic lobes. Western blotting for PCNA confirmed the reduction of cell proliferation by Dox, with the AP and VP being more affected than the dorsal prostate. Thus, Dox treatment alters epithelial cell behavior and prostatic tissue mechanical demand, inducing tissue remodeling in which collagen fibers assume a major role. This work was funded by FAPESP (Sao Paulo State Research Foundation; grants 04/13261-8, to S.L.F.), by FUNDUNESP (Foundation for Development of Sao Paulo State University), and by CNPq (Brazilian National Research and Development Council; fellowship to L.A.J. Jr). This paper represents part of the PhD thesis presented by L.A.J. Jr to the State University of Campinas - UNICAMP, Brazil.     

Regulation of insulin secretion and GLUT4 trafficking by the calcium sensor synaptotagmin VII

Insulin regulates blood glucose by promoting uptake by fat and muscle, and inhibiting production by liver. Insulin-stimulated glucose uptake is mediated by GLUT4, which translocates from an intracellular compartment to the plasma membrane. GLUT4 traffic and insulin secretion both rely on calcium-dependent, regulated exocytosis. Deletion of the voltage-gated potassium channel Kv1.3 results in constitutive expression of GLUT4 at the plasma membrane. Inhibition of channel activity stimulated GLUT4 translocation through a calcium dependent mechanism. The synaptotagmins (Syt) are calcium sensors for vesicular traffic, and Syt VII mediates lysosomal and secretory granule exocytosis. We asked if Syt VII regulates insulin secretion by pancreatic beta cells, and GLUT4 translocation in insulin-sensitive tissues mouse model. Syt VII deletion (Syt VII -/-) results in glucose intolerance and a marked decrease in glucose-stimulated insulin secretion in vivo. Pancreatic islet cells isolated from Syt VII -/- cells secreted significantly less insulin than islets of littermate controls. Syt VII deletion disrupted GLUT4 traffic as evidenced by constitutive expression of GLUT4 present at the plasma membrane of fat and skeletal muscle cells and unresponsiveness to insulin. These data document a key role for Syt VII in peripheral glucose homeostasis through its action on both insulin secretion and GLUT4 traffic.     

Effect of rapid modulation of circulating plasma testosterone concentration on begging,aggressive behavior and competition for food in black-headed gull (Larus ridibundus) chicks

Sibling competition mediated by begging behavior is extremely common in avian species and recent studies have highlighted the role of endogenous testosterone in regulating such phenomenon. However, current literature depicts an inconsistent pattern in altricial vs. semi-precocial species, with stimulating versus inhibitory effects of the hormone respectively. This is possibly due to a difference in the methodology of hormone treatment (short-term moderate dose versus a long-term stronger elevation, respectively) between the studies performed so far. In this study, we induced short-term moderate peaks in plasma testosterone levels, as applied in altricial bird species, and assessed the effects of our manipulation on begging, competitive and aggressive behavior in black-headed gull (Larus ridibundus) chicks, a semi-precocial species. Our results suggest that, unlike in altricial songbirds, temporary increase of plasma testosterone concentration suppresses begging and enhances aggressiveness towards intruders. However, it also increases aggression and the chances of getting priority while scrambling with nest mates to gain access to food. Thus, the inconsistencies in the hormonal control of begging behavior observed between altricial vs. semi-precocial birds seem real and perhaps related to species differences in complexity of the display and the nature of competition. These may be elucidated by future comparative studies.     

Outcomes of testosterone therapy in men with testosterone deficiency (TD): Part II

Testosterone (T) deficiency (TD) is a common clinical condition, which contributes to co-morbidities including loss of muscle mass, increased fat mass, increased inflammation, insulin resistance, risk of vascular disease, sexual dysfunction, fatigue, depressed mood and reduced quality of life. T therapy attenuates inflammation, increases insulin sensitivity, muscle mass and reduces fat mass and adiposity. T therapy improves lipid profiles and endothelial function and reduces systolic and diastolic blood pressure. In addition, T therapy may reduce risk of vascular disease and mortality. T therapy improves bone mineral density and increases energy and vitality and improves mood and sexual function and overall quality of life. T therapy appears to be safe if treatment and monitoring are appropriately executed. The evidence available to date does not support alleged concerns regarding risk of cardiovascular disease and prostate cancer. Indeed, T therapy remains controversial. The data in the contemporary literature suggest that T therapy reduces cardiovascular risk and fears promoted by some recent studies should be re-evaluated. The cardiovascular risk and mortality with T therapy must await large prospective controlled clinical trials, which depend on many complex factors. Such studies may be prohibitive in the current environment due to logistical challenges, such as recruiting large number of men to be treated for long-durations with appropriate follow-up, requiring astronomical cost.     

Caveolin-2 regulation of the cell cycle in response to insulin in Hirc-B fibroblast cells

The regulatory function of caveolin-2 in cell cycle regulation by insulin was investigated in human insulin receptor-overexpressed rat 1 fibroblast (Hirc-B) cells. Insulin increased induction of the caveolin-2 gene in a time-dependent manner. Direct interaction between ERK and caveolin-2 was confirmed by immunoprecipitation and phosphorylated ERK increased the specific interaction in response to insulin. That insulin induced their nuclear co-localization over time was demonstrated by immunofluorescence microscopy. Insulin increased the S phase in the cell cycle by 6-fold. When recombinant caveolin-1 was transiently expressed, a decrease in the S phase was detected by flow-cytometry. The results indicate that the up-regulation of caveolin-2 in response to insulin activates the downstream signal cascades in the cell cycle, chiefly the increased phosphorylation of ERK, the nuclear translocation of phosphorylated ERK, and the subsequent activation of G0/G1 to S phase transition of the cell cycle. The results also suggest that DNA synthesis and the activation of the cell cycle by insulin are achieved concomitantly with an increase in the interaction between caveolin-2 and phosphorylated ERK, and the nuclear translocation of that complex. Taken together, we conclude that caveolin-2 positively regulates the insulin-induced cell cycle through activation of and direct interaction with ERK in Hirc-B cells.     

Stimulation of growth by testosterone via the mesenchyme

Summary To establish testosterone-dependent growth in organ-culture, anlagen of preputial glands from normal wild-type and from androgeninsensitive mouse embryos carrying the testicular feminization mutation (Tfm) were explanted and cultured in the presence of testosterone. Within six days a size difference developed between Tfm and wild-type explants involving length of hair follicle, amount of preputial gland tissue, and overall size.Anlagen from Tfm and wild-type preputial glands were then separated into epithelial bud and mesenchyme. Reciprocal recombinants were prepared and cultured with testosterone. In the recombinants development of hair follicles and gland tissue was inconsistent. Nevertheless, the effect of testosterone was expressed in the overall size of the explants. The size correlated with the type of mesenchyme used, but not with the type of epithelium: Androgen-insensitive Tfm epithelium combined with wild-type mesenchyme reached the same size as whole wild-type glands and wild-type/wild-type recombinants. Wild-type epithelium with Tfm mesenchyme resulted in small explants, which were in the range of the whole androgen-insensitive Tfm glands. Tfm/Tfm recombinants showed very poor growth, probably related to the fact that in this group no hair or gland structures developed.     

Growth factor regulation of cell growth and proliferation in the nervous system

This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crest-derived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.     

Developmental regulation of insulin receptor gene in sciatic nerves and role of insulin on glycoprotein P0 in the Schwann cells

In view of the observations that Schwann cells contain insulin receptors, in the present study, we have investigated the developmental regulation of insulin receptor gene in the sciatic nerves of different postnatal age group rats. We have also investigated the role of insulin in the expression of the major PNS myelin glycoprotein P zero (P0) in normal as well as high glucose conditions in primary rat Schwann cells. The expression of insulin receptor gene in sciatic nerves appeared to be differentially regulated. The steady-state levels of insulin receptor mRNA increased remarkably during development and after postnatal day 10, when the peak of myelin structural gene (P0) expression occur and slowly increased further until at least postnatal day 90 in parallel with the growth of the myelin sheath. By employing immunofluorescence and RT-PCR, we observed significant increase in the P0 protein and mRNA levels in Schwann cells in response to the insulin than in insulin deprived counterparts. The presence of insulin in the high glucose medium ameliorated the altered protein and mRNA of P0 in Schwann cells compared to the insulin deprived counterparts. These studies demonstrate the importance of insulin and its receptor as possible regulatory factors in the PNS and also emphasizes their novel therapeutic applications in demyelinating diseases, especially in diabetic poly-neuropathy.     

睾丸酮对大鼠前列腺上皮细胞有丝分裂方向的影响

目的探讨丙酸睾丸酮（T）对大鼠前列腺上皮细胞染色体有丝分裂方向的影响及其差异基因表达。方法 SPF级SD雄性大鼠（110~130 g）20只,随机分为2组。深麻醉下对所有大鼠进行去势手术,恢复一周后,给药组大鼠皮下注射T,每只0.5 mg,每日1次,连续30d;对照组大鼠皮下注射橄榄油0.1 mL。取前列腺。通过免疫组化、HE染色观察结果。并做基因芯片检查差异基因表达谱。结果给药组大鼠前列腺发生形态学增生。前列腺腺腔扩张,腺上皮高度明显增高,前列腺增生。且前列腺上皮细胞染色体有丝分裂的方向与基底膜平行,而对照组前列腺上皮细胞有丝分裂的方向与基底膜垂直。AR免疫组化染色后发现给药组的前列腺上皮中,均可见到AR标记的阳性细胞,而对照组均为阴性。基因芯片和RT-PCR结果：促进细胞增殖的基因如雄激素受体相关蛋白（RAN）、TGM4和Wnt通道的WNT2等基因均上调,而抑制细胞增殖的基因如负调控Wnt通道的DKK3和促进细胞凋亡的Fas等基因下调。结论 T注射后改变了前列腺上皮细胞有丝分裂的方向,Wnt和AR信号转导通路参与了细胞增殖和有丝分裂方向改变。     

Adventures with insulin in the islets of Langerhans

Insulin is a small but beautifully organized protein with a unique two-chain structure, the first protein to be sequenced. The mechanism of its biosynthesis invited much initial speculation but was finally clarified by the discovery of proinsulin, its single-chain precursor. The rich present-day field of protein precursor processing via post-translational proteolysis within the secretory pathway arose in the early 1970s as an offshoot of studies on insulin biosynthesis, which provided a novel paradigm for the generation of many other small neuroendocrine peptides. Before long, this mechanism was also found to play a role in the production of a much wider spectrum of proteins traversing the secretory pathway (receptors, growth factors, blood-clotting components, and even many viral envelope proteins) occurring in almost all eukaryotic cells. Indeed, yeast provided a key clue in the search for the proprotein convertases, the endoproteases that work along with carboxypeptidases and other modifying enzymes, such as the amidating enzyme complex (PAM), in converting inactive or less active precursor proteins into their fully active peptide products. In this "Reflections" article, I have tried to recount the people and events in my life that led to my involvement first in basic biochemical research and then on to insulin, proinsulin, and many relevant related areas that continue to fascinate and challenge my colleagues and me, as well as many other biomedical scientists today, as diabetes mellitus increasingly threatens human health throughout our contemporary world.