Testicular induced corticosterone synthesis in male rats under fasting stress.

Testicular steroidogenesis is depressed by adrenal-secreted corticosterone (CORT) under stress. However, the mechanisms are not well understood. This study investigated the details of testicular steroidogenesis depression during fasting. Blood levels of adrenocorticotropic hormone secreted from the pituitary glands increased, but blood CORT was not changed in rats that fasted for 96 h, in spite of the rats being severely stressed. CORT in fasting adult male rats increased more than three times in the testis, but reduced testicular testosterone (T) and blood T levels to 5% and 2% of the control, respectively, was observed. The contents of T precursor (except PGN) were drastically reduced in the fasted-rat testes. Testicular CORT levels were elevated, but the enzymatic activity of cytochrome P45011ß, which produces CORT, remained unchanged. The enzymatic activities of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), mediating the conversion of pregnenolone to progesterone, decreased in the fasted-rat testes. Thus, fasting suppressed testicular steroidogenesis by affecting the enzyme activity of 3ß-HSD in the testes and drastically reduced T and increased CORT synthesis. It can be considered that T synthesis involved in cell proliferation is suppressed due to lack of energy during fasting. Conversely, 11ß-hydroxylase enzyme activity was induced and CORT synthesis is increased to cope with the fasting stress. Hence, it can be concluded that CORT synthesis in the testes plays a role in the local defense response.

Circadian gene × environment perturbations influence alcohol drinking in Cryptochrome-deficient mice.

Alcohol use disorder (AUD) is a widespread addiction disorder with severe consequences for health. AUD patients often suffer from sleep disturbances and irregular daily patterns. Conversely, disruptions of circadian rhythms are considered a risk factor for AUD and alcohol relapses. In this study, we investigated the extent to which circadian genetic and environmental disruptions and their interaction alter alcohol drinking behaviour in mice. As a model of genetic circadian disruption, we used Cryptochrome1/2-deficient (Cry1/2-/- ) mice with strongly suppressed circadian rhythms and found that they exhibit significantly reduced preference for alcohol but increased incentive motivation to obtain it. Similarly, we found that low circadian SCN amplitude correlates with reduced alcohol preference in WT mice. Moreover, we show that the low alcohol preference of Cry1/2-/- mice concurs with high corticosterone and low levels of the orexin precursor prepro-orexin and that WT and Cry1/2-/- mice respond differently to alcohol withdrawal. As a model of environmentally induced disruption of circadian rhythms, we exposed mice to a "shift work" light/dark regimen, which also leads to a reduction in their alcohol preference. Interestingly, this effect is even more pronounced when genetic and environmental circadian perturbations interact in Cry1/2-/- mice under "shift work" conditions. In conclusion, our study demonstrates that in mice, disturbances in circadian rhythms have pronounced effects on alcohol consumption as well as on physiological factors and other behaviours associated with AUD and that the interaction between circadian genetic and environmental disturbances further alters alcohol consumption behaviour.

Intergenerational genetic programming mechanism and sex differences of the adrenal corticosterone synthesis dysfunction in offspring induced by prenatal ethanol exposure.

We previously found that prenatal ethanol exposure (PEE) induced adrenal dysplasia in offspring, which was related to intrauterine maternal glucocorticoid overexposure. This study investigated the intergenerational genetic effect and sex differences of PEE-induced changes in the synthetic function of adrenal corticosterone in offspring, and to clarify the intrauterine origin programming mechanism. Wistar pregnant rats were gavaged with ethanol (4 g/kg bw/d) from gestation day (GD) 9-20, and F1 generation was born naturally. The F1 generation female rats in the PEE group were mated with normal male rats to produce F2 generation. Serum and adrenal glands of fetal rats and F1/F2 adult rats were collected at GD20 and postnatal week 28. PEE increased the serum corticosterone level, while diminishing the expression of adrenal steroid synthases of fetal rats. Moreover, PEE enhanced the mRNA expression of GR and HDAC1, but inhibited the mRNA expression of SF1 and reduced the H3K9ac level of P450scc in the fetal adrenal gland. In PEE adult offspring of F1 and F2 generation the serum corticosterone level, the H3K9ac level of P450scc and its expression were decreased in males but were increased in females. In NCI-H295R cells, cortisol reduced the production of endogenous cortisol, down-regulated SF1, and up-regulated HDAC1 expression by activating GR, and decreased H3K9ac level and expression of P450scc. In conclusion, PEE could induce adrenal dysplasia in offspring with sex differences and intergenerational genetic effects, and the adrenal insufficiency in male offspring was related to the induction of low functional genetic programming of P450scc by intrauterine high corticosterone through the GR/SF1/HDAC1 pathway.

Isoflurane stress induces glucocorticoid production in mouse lymphoid organs.

Glucocorticoids (GCs) are secreted by the adrenal glands and locally produced by lymphoid organs. Adrenal GC secretion at baseline and in response to stressors is greatly reduced during the stress hyporesponsive period (SHRP) in neonatal mice (postnatal day (PND) 2-12). It is unknown whether lymphoid GC production increases in response to stressors during the SHRP. Here, we administered an acute stressor (isoflurane anesthesia) to mice before, during, and after the SHRP and measured systemic and local GCs via mass spectrometry. We administered isoflurane, vehicle control (oxygen), or neither (baseline) at PND 1, 5, 9, or 13 and measured progesterone and six GCs in blood, bone marrow, thymus, and spleen. At PND1, blood and lymphoid GC levels were high and did not respond to stress. At PND5, blood GC levels were very low and increased slightly after stress, while lymphoid GC levels were also low but increased greatly after stress. At PND9, blood and lymphoid GC levels were similar at baseline and increased similarly after stress. At PND13, blood GC levels were higher than lymphoid GC levels at baseline, and blood GC levels showed a greater response to stress. These data demonstrate the remarkable plasticity of GC physiology during the postnatal period, show that local steroid levels do not reflect systemic steroid levels, provide insight into the SHRP, and identify a potential mechanism by which early-life stressors can alter immunity in adulthood.

Intrauterine growth restriction leads to a high-corticosterone producing offspring: An implication for pulmonary infection susceptibility.

AIMS: Although intrauterine growth restriction (IUGR) impairs immune system homeostasis and lung development, its relationship with the susceptibility to pulmonary infections remains unclear. Thus, this study aimed to investigate the impact of IUGR on acute lung inflammatory response induced by bacterial stimulus. MATERIALS AND METHODS: Pregnant female Wistar rats were subjected to 50% caloric-protein food restriction during gestation. To mimic bacterial lung infection, adult male offspring (12 weeks old) were challenged with a single lipopolysaccharide (LPS) intranasal instillation, and 6 h later, we assessed the acute inflammatory response. Normal birth weight (NBW) animals represent the control group. KEY FINDINGS: LPS instillation increased the protein levels in the airways of both the NBW and low birth weight (LBW) groups, indicating vascular leakage. LBW animals exhibited a lower number of neutrophils, reduced production of interleukin-6 and macrophage-inflammatory protein-2 and decreased upregulation of intercellular adhesion molecule-1 gene expression in lung tissues. Further analysis revealed that the LBW group produced lower levels of prostaglandin-E2 and failed to secrete leukotriene-B4 upon LPS stimulation, which correlated with impaired cyclooxygenase-2 and 5-lipoxygenase expression. These results were probably associated with their inability to upregulate the expression of Toll-like receptor-4 and downstream signaling proteins, such as nuclear factor kappa-B, in the lungs. The LBW group also exhibited abnormal airway thickening and high corticosterone levels under basal conditions. SIGNIFICANCE: This study suggests that IUGR-induced foetal programming in LBW offspring threatens HPA axis physiology and corticosterone biodisponibility, and impairs the innate response to bacterial antigens, increasing future susceptibility to pulmonary infection.

Psychosocial Crowding Stress-Induced Changes in Synaptic Transmission and Glutamate Receptor Expression in the Rat Frontal Cortex.

This study demonstrates how exposure to psychosocial crowding stress (CS) for 3, 7, and 14 days affects glutamate synapse functioning and signal transduction in the frontal cortex (FC) of rats. CS effects on synaptic activity were evaluated in FC slices of the primary motor cortex (M1) by measuring field potential (FP) amplitude, paired-pulse ratio (PPR), and long-term potentiation (LTP). Protein expression of GluA1, GluN2B mGluR1a/5, VGLUT1, and VGLUT2 was assessed in FC by western blot. The body's response to CS was evaluated by measuring body weight and the plasma level of plasma corticosterone (CORT), adrenocorticotropic hormone (ACTH), and interleukin 1 beta (IL1B). CS 3 14d increased FP and attenuated LTP in M1, while PPR was augmented in CS 14d. The expression of GluA1, GluN2B, and mGluR1a/5 was up-regulated in CS 3d and downregulated in CS 14d. VGLUTs expression tended to increase in CS 7d. The failure to blunt the effects of chronic CS on FP and LTP in M1 suggests the impairment of habituation mechanisms by psychosocial stressors. PPR augmented by chronic CS with increased VGLUTs level in the CS 7d indicates that prolonged CS exposure changed presynaptic signaling within the FC. The CS bidirectional profile of changes in glutamate receptors' expression seems to be a common mechanism evoked by stress in the FC.

Effects of ChREBP deficiency on adrenal lipogenesis and steroidogenesis.

Carbohydrate response element-binding protein (ChREBP) is critical in the regulation of fatty acid and triglyceride synthesis in the liver. Interestingly, Chrebp-/- mice show reduced levels of plasma cholesterol, which is critical for steroid hormone synthesis in adrenal glands. Furthermore, Chrebp mRNA expression was previously reported in human adrenal glands. Thus, it remains to be investigated whether ChREBP plays a role directly or indirectly in steroid hormone synthesis and release in adrenal glands. In the present study, we find that Chrebp mRNA is expressed in mouse adrenal glands and that ChREBP binds to carbohydrate response elements. Histological analysis of Chrebp-/- mice shows no adrenal hyperplasia and less oil red O staining compared with that in WT mice. In adrenal glands of Chrebp-/- mice, expression of Fasn and Scd1, two enzymes critical for fatty acid synthesis, was substantially lower and triglyceride content was reduced. Expression of Srebf2, a key transcription factor controlling synthesis and uptake of cholesterol and the target genes, was upregulated, while cholesterol content was not significantly altered in the adrenal glands of Chrebp-/- mice. Adrenal corticosterone content and plasma adrenocorticotropic hormone and corticosterone levels were not significantly altered in Chrebp-/- mice. Consistently, expression of genes related to steroid hormone synthesis was not altered. Corticosterone secretion in response to two different stimuli, namely 24-h starvation and cosyntropin administration, was also not altered in Chrebp-/- mice. Taking these results together, corticosterone synthesis and release were not affected in Chrebp-/- mice despite reduced plasma cholesterol levels.

Corticosterone Is Essential for Survival Through Frog Metamorphosis.

Thyroid hormone (TH) is required for frog metamorphosis, and corticosterone (CORT) increases TH signaling to accelerate metamorphic progression. However, a requirement for CORT in metamorphosis has been difficult to assess prior to the recent development of gene-editing technologies. We addressed this long-standing question using transcription activator-like effector nuclease (TALEN) gene disruption to knock out proopiomelanocortin (pomc) and disrupt CORT production in Xenopus tropicalis. As expected, mutant tadpoles had a reduced peak of plasma CORT at metamorphosis with correspondingly reduced expression of the CORT-response gene Usher syndrome type-1G (ush1g). Mutants had reduced rates of growth and development and exhibited lower expression levels of 2 TH response genes, Krüppel-like factor 9 (klf9) and TH receptor ß (thrb). In response to exogenous TH, mutants had reduced TH response gene induction and slower morphological change. Importantly, death invariably occurred during tail resorption, unless rescued by exogenous CORT and, remarkably, by exogenous TH. The ability of exogenous TH by itself to overcome death in pomc mutants indicates that the CORT-dependent increase in TH signaling may ensure functional organ transformation required for survival through metamorphosis and/or may shorten the nonfeeding metamorphic transition to avoid lethal inanition.

Local biosynthesis of corticosterone in rat skeletal muscle.

Adrenal corticosterone plays crucial roles in energy metabolism and immuno-reactivity throughout the body. As we have previously shown that corticosterone biosynthesis in C2C12 myoblasts, we study about corticosterone biosynthesis in rat skeletal muscles. It was found that enzymatic activities producing corticosterone and testosterone except the activity of P450scc in rat skeletal muscle as like as C2C12 cells. The CYP11B mRNA encoding cytochrome P45011ß that mediates 11-deoxycorticosterone hydroxylase activity, producing corticosterone was expressed in skeletal muscles. In immunoblotting analysis, cytochrome P45011ß protein was expressed in rat muscles and whole organs especially higher levels in adrenal and brain. The localizations of corticosterone content and enzymatic activities involved in the production of corticosterone were preferentially observed in gastrocnemius fibers rather than in soleus fibers. The immunohistochemical analysis showed that the fast-twitch or type II muscle fibers positive to antibody against fast myosin heavy chain were preferentially stained with anti-cytochrome P45011ß antibody in the gastrocnemius fiber. In addition, we detected corticosterone biosynthesis from pregnenolone sulfate conjugates in perfusion of the rat hindquarter. Corticosterone is synthesized in rat skeletal muscles and the biosynthesis was localized in the fast-twitch or type II muscle fibers. We speculated that the local synthesized corticosterone might be involved in glucocorticoid-induced muscle atrophy that preferentially occurs in fast muscle fibers, and the initial substrate of the local CORT biosynthesis were supported to be performed from the conjugates such as pregnenolone sulfate circulating in the blood flow.

Intestinal Flora Modulates Blood Pressure by Regulating the Synthesis of Intestinal-Derived Corticosterone in High Salt-Induced Hypertension.

RATIONALE: High-salt diet is one of the most important risk factors for hypertension. Intestinal flora has been reported to be associated with high salt-induced hypertension (hSIH). However, the detailed roles of intestinal flora in hSIH pathogenesis have not yet been fully elucidated. OBJECTIVE: To reveal the roles and mechanisms of intestinal flora in hSIH development. METHODS AND RESULTS: The abovementioned issues were investigated using various techniques including 16S rRNA gene sequencing, untargeted metabolomics, selective bacterial culture, and fecal microbiota transplantation. We found that high-salt diet induced hypertension in Wistar rats. The fecal microbiota of healthy rats could dramatically lower blood pressure (BP) of hypertensive rats, whereas the fecal microbiota of hSIH rats had opposite effects. The composition, metabolism, and interrelationship of intestinal flora in hSIH rats were considerably reshaped, including the increased corticosterone level and reduced Bacteroides and arachidonic acid levels, which tightly correlated with BP. The serum corticosterone level was also significantly increased in rats with hSIH. Furthermore, the above abnormalities were confirmed in patients with hypertension. The intestinal Bacteroides fragilis could inhibit the production of intestinal-derived corticosterone induced by high-salt diet through its metabolite arachidonic acid. CONCLUSIONS: hSIH could be transferred by fecal microbiota transplantation, indicating the pivotal roles of intestinal flora in hSIH development. High-salt diet reduced the levels of B fragilis and arachidonic acid in the intestine, which increased intestinal-derived corticosterone production and corticosterone levels in serum and intestine, thereby promoting BP elevation. This study revealed a novel mechanism different from inflammation/immunity by which intestinal flora regulated BP, namely intestinal flora could modulate BP by affecting steroid hormone levels. These findings enriched the understanding of the function of intestinal flora and its effects on hypertension.

Royal jelly reduces depression-like behavior through possible effects on adrenal steroidogenesis in a murine model of unpredictable chronic mild stress.

Royal jelly (RJ) is used as a dietary supplement for human health promotion. Recently, a clinical trial has reported that RJ improved mental health. The present study was conducted to experimentally support the clinical effect of RJ on mental health and to further elucidate the mechanisms of action of RJ. RJ and an ethanol extract of RJ, which contains fatty acids but not proteins, inhibited an unpredictable chronic mild stress (UCMS)-induced increase in immobility time, a depression-like behavior, in the tail suspension test. DNA microarray analysis of the adrenal grand revealed that the expression of genes involved in cholesterol metabolism was up-regulated in response to UCMS exposure and that RJ suppressed expression of genes related to cholesterol synthesis and transport. These results suggested that RJ improves stress-induced depression-like behavior by regulating adrenal steroidogenesis and that fatty acids contained in RJ partly contribute to the antidepressant effect of RJ.

Corticosteroid-Binding Globulin is expressed in the adrenal gland and its absence impairs corticosterone synthesis and secretion in a sex-dependent manner.

Corticosteroid-binding globulin (CBG) is synthesized by the liver and secreted into the bloodstream where binds to glucocorticoids. Thus CBG has the role of glucocorticoid transport and free hormone control. In addition, CBG has been detected in some extrahepatic tissues without a known role. CBG-deficient mice show decreased total corticosterone levels with missing of classical sexual dimorphism, increased free corticosterone, higher adrenal gland size and altered HPA axis response to stress. Our aim was to ascertain whether CBG deficiency could affect the endocrine synthetic activity of adrenal gland and if the adrenal gland produces CBG. We determined the expression in adrenal gland of proteins involved in the cholesterol uptake and its transport to mitochondria and the main enzymes involved in the corticosterone, aldosterone and catecholamine synthesis. The results showed that CBG is synthesized in the adrenal gland. CBG-deficiency reduced the expression of ACTH receptor, SRB1 and the main genes involved in the adrenal hormones synthesis, stronger in females resulting in the loss of sexual dimorphism in corticosteroid adrenal synthesis, despite corticosterone content in adrenal glands from CBG-deficient females was similar to wildtype ones. In conclusion, these results point to an unexplored and relevant role of CBG in the adrenal gland functionality related to corticosterone production and release.

Topically injected adrenocorticotropic hormone induces mechanical hypersensitivity on a full-thickness cutaneous wound model in rats.

Cutaneous wound pain causes physical and psychological stress for patients with wounds. Previous studies reported that stress induces hyperalgesia and deteriorates wound healing. However, the effect of the stress response such as in hypothalamic-pituitary-adrenal (HPA) axis on local wound area is unclear. We aimed to investigate the effects of a stress response on the mechanical withdrawal threshold in the local wound area and describe the identification of a wound pain exacerbation. We topically injected adrenocorticotropic hormone (ACTH) into the granulation tissue of full-thickness cutaneous wound model rats on the fifth day postwounding and measured the mechanical withdrawal thresholds, cytochrome P450 2Bs levels and concentration of 5,6-epoxyeicosatrienoic acid in wound exudate. We found that ACTH induced mechanical hypersensitivity at 4 and 6 hours after injection (P = .004 and .021, respectively), and increased gene expression of cytochrome P450 2B12 expression (P = .046). Concentration of 5,6-EET in the wound exudate was moderately correlated with the mechanical withdrawal threshold (r = -.630). Finally, the mechanical withdrawal threshold in the 5,6-EET group was significantly lower than that in the control group at 2 hours after the injection (P = .015). We propose that 5,6-EET is one of the most promising contributors to the wound pain exacerbation. These findings could guide clinical wound and pain management.

Capsaicin inhibits lipopolysaccharide-induced adrenal steroidogenesis by raising intracellular calcium levels.

INTRODUCTION: Glucocorticoid release by adrenals has been described as significant to survive sepsis. The activation of transient receptor potential vanilloid type 1 (TRPV1) inhibited ACTH-induced glucocorticoid release by adrenal glands in vitro. OBJECTIVE: The aim of this study was to investigate if capsaicin, an activator of TRPV1, would prevent LPS-induced glucocorticoid production by adrenals. METHODS: Male Swiss-Webster mice were treated with capsaicin intraperitoneally (0.2 or 2 mg/kg) 30 min before LPS injection. All analyses were performed 2 h after the LPS stimulation, including plasma corticosterone and peritoneal IL-1ß and TNF-α levels. Furthermore, murine adrenocortical Y1 cells were used to assess the effects of capsaicin on LPS-induced corticosterone production in vitro. RESULTS: Capsaicin (2 mg/kg, i.p.) significantly reduced plasma corticosterone levels and adrenal hypertrophy induced by LPS without alter the levels of pro-steroidogenic cytokines IL-1ß and TNF-α in peritoneal cavity of mice, while the dose of 0.2 mg/kg of capsaicin did not interfere with adrenal steroidogenesis, attested by RIA and ELISA, respectively. Y1 cells express TRPV1, measured by immunofluorescence and western blot, and capsaicin decreased LPS-induced corticosterone production by these cells in vitro. Capsaicin also induces calcium mobilization in Y1 cells in vitro. CONCLUSIONS: These findings suggest that capsaicin inhibits corticosterone production induced by LPS by acting directly on adrenal cells producing glucocorticoids, in a mechanism probably associated with induction of a cytoplasmic calcium increase in these cells.

Infradian Rhythms of Resistance to a Dissociative Anesthetic in Wistar Male Rats under Normal Conditions and After Surgical Removal of the Adrenal Glands and Testes.

Daily dynamics of changes in the latency of a response to dissociative anesthetic tiletamine (time from injection to ataxia) was studied in mature Wistar rats. Both intramuscular and intravenous administration of the anesthetic was associated with 4-day oscillations of the latent period synchronous with the dynamics of changes in the concentration of glucocorticoid hormones. The period and phases of the infradian rhythm of resistance to the anesthetic remained unchanged after removal of both adrenal glands and testes and administration of corticosterone synthesis blocker trilostane diminishing the 4-day cycle of changes in corticosterone level. Therefore, hormones of the adrenal glands and testes do not play the key role in the mechanisms of formation of the 4-day infradian rhythm.

Insulin Regulates Adrenal Steroidogenesis by Stabilizing SF-1 Activity.

Development of metabolic syndrome is associated with hyperactivity of the HPA axis characterized by elevated levels of circulating adrenal hormones including cortisol and aldosterone. However, the molecular mechanism leading to the dysregulation of the HPA axis is not well elucidated. In this study, we found that insulin regulates adrenal steroidogenesis by increasing the expression and activity of steroidogenic factor 1 (SF-1) both in vitro and in vivo and this insulin effect was partly through inhibition of FoxO1. Specifically, insulin increased the protein and RNA levels of SF-1 and steroidogenic target genes. Further, adrenal SF-1 expression was significantly increased by hyperactivation of insulin signaling in mice. Together with the elevated SF-1 expression in adrenal glands, hyperactivation of insulin signaling led to increased aldosterone and corticosterone levels. On the other hand, suppressing the insulin signaling using streptozotocin markedly reduced the expression of adrenal SF-1 in mice. In addition, overexpression of FoxO1 significantly suppressed SF-1 and its steroidogenic target genes implying that the positive effect of insulin on SF-1 activity might be through suppression of FoxO1 in the adrenal gland. Taken together, these results indicate that insulin regulates adrenal steroidogenesis through coordinated control of SF-1 and FoxO1.

Inhibition of corticosterone synthesis and its effect on acute phase proteins, heat shock protein 70, and interleukin-6 in broiler chickens subjected to feed restriction.

The aim of the current study was to elucidate whether inhibition of corticosterone (CORT) synthesis could modify stress response to feed deprivation and its possible interactions with feed restriction in the neonatal period in broiler chickens. Equal numbers of broiler chicks were subjected to either 60% feed restriction (60FR) or ad libitum (AL) on d 4, 5, and 6. On day 7, blood CORT, acute phase proteins (APP), interleukin-6 (IL-6) levels, and brain heat shock protein 70 (HSP70) expression were determined. On d 35, chickens in each early age feeding regimen were subjected to one of the following treatments: (i) ad libitum feeding (ALF), (ii) 24 h feed deprivation (SFR), or (iii) 24 h feed deprivation with intramuscular injection of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) at 100 mg/kg BW (SFR+DDT). The effect of SFR on CORT, APP, IL-6, and HSP 70 were determined on d 36. The results showed that subjecting chicks to 60FR significantly elevated CORT and brain HSP70 concentration compared to the AL group on d 7. The early feeding regimen had no significant effect on CORT, alpha-1 acid glycoprotein (AGP), ovotransferrin (OVT), ceruoplasmin (CP), IL-6, or brain HSP70 on d 36. The CORT, AGP, OVT, CP, IL-6, and brain HSP70 expression of SFR birds following 24 h of feed deprivation (d 36) were significantly higher than their ALF and SFR+DDT counterparts. Both ALF and SFR+DDT birds had similar values. Stress attributed to feed deprivation without concurrent increase in CORT had a negligible effect on serum levels of APP and IL-6 and brain HSP70 expression.

Steroidogenic abnormalities in translocator protein knockout mice and significance in the aging male.

The translocator protein (TSPO) has been proposed to act as a key component in a complex important for mitochondrial cholesterol importation, which is the rate-limiting step in steroid hormone synthesis. However, TSPO function in steroidogenesis has recently been challenged by the development of TSPO knockout (TSPO-KO) mice, as they exhibit normal baseline gonadal testosterone and adrenal corticosteroid production. Here, we demonstrate that despite normal androgen levels in young male TSPO-KO mice, TSPO deficiency alters steroidogenic flux and results in reduced total steroidogenic output. Specific reductions in the levels of progesterone and corticosterone as well as age-dependent androgen deficiency were observed in both young and aged male TSPO-KO mice. Collectively, these findings indicate that while TSPO is not critical for achieving baseline testicular and adrenal steroidogenesis, either indirect effects of TSPO on steroidogenic processes, or compensatory mechanisms and functional redundancy, lead to subtle steroidogenic abnormalities which become exacerbated with aging.

Usefulness of Simultaneous Measurement of Plasma Steroids, Including Precursors, for the Evaluation of Drug Effects on Adrenal Steroidogenesis in Rats.

The aim of this study was to evaluate the usefulness of simultaneous measurement of plasma steroids, including precursors, for the evaluation of drug effects on adrenal steroidogenesis in vivo. Plasma concentrations of corticosterone and its precursors were examined in rats dosed with compounds that affect adrenal steroidogenesis via different modes of action as well as the relationships of the changes with blood chemistry and adrenal histopathology. Male rats were dosed with tricresyl phosphate, aminoglutethimide, trilostane (TRL), metyrapone (MET), ketoconazole (KET), or mifepristone for 7 days. In the TRL, MET, and KET groups, precursor levels were markedly increased, while there were no significant changes in the corticosterone level, suggesting that the precursors are more sensitive biomarkers to detect the effect on adrenal steroidogenesis. Also, the precursors with increased levels were those that are normally metabolized by the inhibited enzymes, reflecting the modes of action of the compounds. In addition, different patterns of changes were observed in blood chemistry and histopathology, supporting the mechanism suggested by the steroid changes. These results show that simultaneous measurement of plasma steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.

TSPO mutations in rats and a human polymorphism impair the rate of steroid synthesis.

The 18 kDa translocator protein (TSPO) is a ubiquitous conserved outer mitochondrial membrane protein implicated in numerous cell and tissue functions, including steroid hormone biosynthesis, respiration, cell proliferation, and apoptosis. TSPO binds with high affinity to cholesterol and numerous compounds, is expressed at high levels in steroid-synthesizing tissues, and mediates cholesterol import into mitochondria, which is the rate-limiting step in steroid formation. In humans, the rs6971 polymorphism on the TSPO gene leads to an amino acid substitution in the fifth transmembrane loop of the protein, which is where the cholesterol-binding domain of TSPO is located, and this polymorphism has been associated with anxiety-related disorders. However, recent knockout mouse models have provided inconsistent conclusions of whether TSPO is directly involved in steroid synthesis. In this report, we show that TSPO deletion mutations in rat and its corresponding rs6971 polymorphism in humans alter adrenocorticotropic hormone-induced plasma corticosteroid concentrations. Rat tissues examined show increased cholesteryl ester accumulation, and neurosteroid formation was undetectable in homozygous rats. These results also support a role for TSPO ligands in diseases with steroid-dependent stress and anxiety elements.