Fu-Kong Lieu

Role of testosterone in regulating induction of TNF-α in rat spleen via ERK signaling pathway.

Spleen is a pivotal organ for regulating immune homeostasis. It has been shown that testosterone diminishes secretion of various inflammatory molecules under multiple conditions. However, the mechanisms of action of endogenous testosterone affecting immune responses in the spleen remain unknown. The aim of the present study was to evaluate the immune functions of the spleen in response to testosterone withdrawal after orchidectomy, and the impact of splenocytes on the bacterial endotoxin lipopolysaccharide (LPS)-induced secretion of inflammatory molecules. Male rats were divided into 3 groups, i.e. intact, orchidectomized (Orch) and orchidectomized plus replacement of testosterone propionate (TP) (Orch+TP). The Orch and Orch+TP rats underwent bilateral orchidectomy one week before TP replacement (2mg/kg body weight) or sesame oil in intact rats as controls for seven days. Orch resulted in a significant increase of spleen weight and basal secretion of nitric oxide (NO) from splenocytes. Additionally, LPS up-regulated cell proliferation and the secretion of tumor necrosis factor-alpha (TNF-α) in splenocytes of Orch rats. Orch further up-regulated phosphorylation of extracellular signal-regulated kinases. Interestingly, the plasma corticosterone concentration in the Orch group was higher than that in the intact and Orch+TP groups. Deficiency of testosterone-elevated TNF-α and NO secretion in response to LPS were confirmed in the rat splenocytes. Testosterone also significantly attenuated LPS-elicited release of TNF-α and NO in a dose-dependent manner. However, testosterone did not suppress splenic blastogenesis at doses in the 10(-10)-10(-7)M concentration range. In this context, testosterone might have a protective role against inflammatory responses in the spleen. The present study provides evidence to indicate that testosterone might modulate the immune system.

Interactive Effect of Corticosterone and Lactate on Regulation of Testosterone Production in Rat Leydig Cells

The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5–20 mM), cortiosterone (10−9–10−4 M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side‐chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate‐stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135–2144, 2017.

Effects of acrolein on aldosterone release from zona glomerulosa cells in male rats

A positive correlation between smoking and hypertension has been well established. Acrolein is a major toxic volatile compound found in cigarette smoke. Human exposure to low levels of acrolein is unavoidable due to its production in daily activities, such as smoke from industrial, hot oil cooking vapors, and exhaust fumes from vehicles. The toxicity and the action mechanism of acrolein to induce apoptosis have been extensively studied, but the effects of acrolein on hypertension are still unknown. The present study aimed to examine the effects of acrolein on aldosterone release both in vivo and in vitro. Male rats were divided into three groups, and intraperitoneally injected with normal saline, or acrolein (2mg/kg) for 1 (group A-1) or 3 (group A-3) days, respectively. After sacrificing, rat blood samples were obtained to measure plasma aldosterone and angiotensin II (Ang II) levels. Zona glomerulosa (ZG) cells were prepared from rat adrenal cortex, and were incubated with or without stimulants. We found that the serum aldosterone was increased by 1.2-fold (p<0.05) in A-3 group as compared to control group. Basal aldosterone release from ZG cells in A-3 group was also increased significantly. Moreover, acrolein enhanced the stimulatory effects of Ang II and 8-bromo-cyclic AMP on aldosterone secretion from ZG cells prepared in both A-1 and A-3 groups. Furthermore, the enzyme activity of P450scc, the rate-limiting step of aldosterone synthesis, was elevated after acrolein injection. Plasma level of Ang II was increased in both A-1 and A-3 groups. These results suggested that acrolein exposure increased aldosterone production, at least in part, through elevating the level of plasma Ang II and stimulating steroidogenesis pathways.

Attenuation of exercise effect on inflammatory responses via novel role of TLR4/PI3K/Akt signaling in rat splenocytes

Moderate exercise diminishes proinflammation cytokine production in various types of immune cells, but the intracellular signaling pathways involved are not completely understood. Phosphoinositide 3-kinase (PI3K)/Akt, a crucial downstream protein of toll-like receptor 4 (TLR4), may modulate inflammation. The present study aimed to investigate the effects of exercises on lipopolysaccharide (LPS)-stimulated inflammatory response in splenocytes and to explore potential mechanisms of the PI3K/Akt pathway. Male rats were divided into sedentary and exercise groups. Animals in the exercise group underwent endurance training 30 min/day, 7 days/wk, at the speed of 20 m/min on a treadmill for 1 wk. Here, we showed that exercise 1) attenuated TLR4, 2) increased PI3K/phospho-Akt (p-Akt), and 3) diminished phospho-nuclear factor-κB (p-NF-κB) expression. In addition, administration of splenocytes isolated from trained rats with LPS in vitro showed 1) reduced tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and nitric oxide secretion and 2) decreased splenocyte proliferation. The plasma corticosterone (CCS) level in the exercise group was higher than that in the sedentary group. We confirmed that CCS down-regulated TNF-α and IL-6 secretion in response to LPS in rat splenocytes. Dexamethasone also significantly attenuated LPS-evoked release of TNF-α and IL-6 in a dose-dependent manner. These findings suggested that exercise dampened the secretion of inflammation mediators probably through partial inhibition of TLR4 and p-NF-κB and activation of PI3K/p-Akt expression in the spleen.

Effects of acrolein on the production of corticosterone in male rats

Acrolein, an α, β-unsaturated aldehyde, exists in a wide range of sources. Acrolein can be not only generated from all types of smoke but also produced endogenously from the metabolism by lipid peroxidation. The cellular influence of acrolein is due to its electrophilic character via binding to and depleting cellular nucleophiles. Although the toxicity of acrolein has been extensively studied, there is relatively little information about its impact on hormone release. This study aimed at the effect of acrolein on hypothalamic-pituitary-adrenal (H-P-A) axis. In an in vivo study, male rats were administrated with acrolein for 1 or 3days. The plasma corticosterone in response to a single injection of adrenocorticotropic hormone (ACTH) increased slowly in acrolein-pretreated rats than in control rats. Further investigating the steroidogenic pathway, the protein expressions of steroidogenic acute regulatory protein (StAR) and the upper receptor-melanocortin 2 receptor (MC2R) were attenuated in acrolein-treated groups. Another experiment using trilostane showed less activity of P450scc in zona fasciculata-reticularis (ZFR) cells in acrolein-treated groups. In addition to the suppressed ability of corticosterone production in ZFR cells, acrolein even had extended influence at higher concentrations. The lower ACTH was observed in the plasma from acrolein-pretreated rats. In an in vitro study, ZFR cells were incubated with acrolein and the results showed that corticosterone concentrations in media were decreased in a dose-dependent manner. Acrolein also desensitized the response of the ZFR cells to ACTH. These results suggested that acrolein decreased the releasing ability of corticosterone via an inhibition on the response of ZFR cells to ACTH and the reduction of protein expressions of StAR and MC2R as well as the activity of P450scc in rat ZFR cells. The present evidences showed that the H-P-A axis was affected by the administration of acrolein.

Adaptation of Testosterone Production in Response to Low-Dose Effects of Nonylphenol

Nonylphenol (NP) is a non-ionic surfactant that is used widely as an industrial detergent. We have found that NP impairs male reproductive functions in vitro. However, the in vivo effects of NP on testosterone release at environmentally relevant doses remain unclear. Male rats were exposed to a low-dose of NP by oral gavage at 10 and 100 μg/kg body weight daily for 3-7 days. After 7 days of NP exposure, plasma testosterone level was significantly higher in higher NP exposure group than in the vehicle group. Higher basal testosterone release was found in the Leydig cells obtained from the rats treated with NP for 7 days. Additionally, evoked testosterone release was also higher after treating Leydig cells with human chorionicgonadotropin, 8-bromo-adenosine cyclic monophosphate, or forskolin. The expression of steroidogenic acute regulatory protein (StAR) was also increased by 40% in oral NP group. In contrast, the stimulatory effects were absent in Leydig cells treated with various steroid precursors. The stimulatory effects of NP were abolished after administration with intracellular calcium, EGFR and ERK blockers, which were well known to stimulate StAR activation. These results suggest that NP at an environmentally relevant dose could stimulate plasma testosterone level.

Effect of Swimming on the Production of Aldosterone in Rats

It has been demonstrated that exercise is one of the stresses known to increase the aldosterone secretion. Both potassium and angiotensin II (Ang II) levels are shown to be correlated with aldosterone production during exercise, but the mechanism is still unclear. In an in vivo study, male rats were catheterized via right jugular vein (RJV), and divided into four groups namely water immersion, swimming, lactate infusion (13 mg/kg/min) and pyruvate infusion (13 mg/kg/min) groups. Each group was treated for 10 min. Blood samples were collected at 0, 10, 15, 30, 60 and 120 min from RJV after administration. In an in vitro study, rat zona glomerulosa (ZG) cells were challenged by lactate (1–10 mM) in the presence or absence of Ang II (10−8 M) for 60 min. The levels of aldosterone in plasma and medium were measured by radioimmunoassay. Cell lysates were analyzed by immunoblotting assay. After exercise and lactate infusion, plasma levels of aldosterone and lactate were significantly higher than those in the control group. Swimming for 10 min significantly increased the plasma Ang II levels in male rats. Administration of lactate plus Ang II significantly increased aldosterone production and enhanced protein expression of steroidogenic acute regulatory protein (StAR) in ZG cells. These results demonstrated that acute exercise led to the increase of both aldosterone and Ang II secretion, which is associated with lactate action on ZG cells and might be dependent on the activity of renin-angiotensin system.

Stimulatory Effect of Intermittent Hypoxia on the Production of Corticosterone by Zona Fasciculata-Reticularis Cells in Rats

Hypoxia or intermittent hypoxia (IH) have known to alter both synthesis and secretion of hormones. However, the effect of IH on the production of adrenal cortical steroid hormones is still unclear. The aim of present study was to explore the mechanism involved in the effect of IH on the production of corticosterone by rat ZFR cells. Male rats were exposed at 12% O2 and 88% N2 (8 hours per day) for 1, 2, or 4 days. The ZFR cells were incubated at 37 °C for 1 hour with or without ACTH, 8-Br-cAMP, calcium ion channel blockers, or steroidogenic precursors. The concentration of plasma corticosterone was increased time-dependently by administration of IH hypoxia. The basal levels of corticosterone production in cells were higher in the IH groups than in normoxic group. IH resulted in a time-dependent increase of corticosterone production in response to ACTH, 8-Br-cAMP, progesterone and deoxycorticosterone. The production of pregnenolone in response to 25-OH-C and that of progesterone in response to pregnenolone in ZFR cells were enhanced by 4-day IH. These results suggest that IH in rats increases the secretion of corticosterone via a mechanism at least in part associated with the activation of cAMP pathway and steroidogenic enzymes.

Induction of renal senescence marker protein-30 (SMP30) expression by testosterone and its contribution to urinary calcium absorption in male rats.

The aim of this study was to investigate the involvement of androgen, mainly testosterone, in the expression of renal senescence marker protein-30 (SMP30) in male rats. We found that the renal SMP30 expression was up-regulated by endogenous testosterone stimulation during puberty. Interestingly, androgen-deficient orchidectomized (ORX) rats exhibited lower SMP30 mRNA and protein expression in the kidney, and that was restored by testosterone propionate (TP) replacement. Abrogation of androgen receptor (AR) activity by co-treatment with flutamide abolished testosterone-induced SMP30 expression in the kidney as well as in the NRK52E cells. However, SMP30 expression was unaltered in the liver of ORX rats. We also showed a positive correlation between renal SMP30 expression and plasma testosterone level during the aging process. TP-induced SMP30 expression in ovariectomized (OVX) rats was observed and was an evidence to explain the gender difference of SMP30 levels. Immunofluorescence assay showed that renal SMP30 was specifically expressed in the proximal tubular segments of the kidney. The urinary Ca2+ level was increased in both ORX and male aging rats. Taken together, our results indicate a novel role of testosterone in regulating SMP30 expression specifically in the kidney to contribute to urinary calcium absorption.