Mechanism of skeletal muscle atrophy by muscle fiber types in male rats under long-term fasting stress.

Fasting induces metabolic changes in muscles, which are differentiated by muscle fiber type. In this study, the mechanism of fasting-induced muscle atrophy in rats was examined to determine the differences between muscle fiber types in energy production. Fasting for 96 h did not alter the weight of the soleus (SOL), a fiber type I muscle, but did significantly reduce the weight of gastrocnemius (GM), a fiber type II muscle. GM, SOL and blood pregnenolone and testosterone levels decreased under fasting, which induced energy deprivation, whereas corticosterone (CORT) levels significantly increased. However, the expression of 3ß-HSD and P45011ß in GM was unaffected by fasting. The decrease in GM weight may be due to decreased levels of testosterone and reduced synthesis of mammalian target of rapamycin (mTOR). Significant increases in CORT both GM and SOL were associated with increases in the amount of branched-chain amino acids available for energy production. However, decreased levels of mTOR and IGF1 and increased levels of CORT and IL-6 in SOL suggest that GM proteolysis was followed by SOL proteolysis for additional energy production. In conclusion, IGF1 levels decreased significantly in SOL, whereas those of IL-6 significantly increased in SOL and blood but decreased in GM. Blood branched-chain amino acids (BCAA) levels were unaffected due to fasting, whereas an increase was noted in the levels of BCAA in GM and SOL. These results show that fasting for 96 h restricts energy supply, producing fast-twitch muscle atrophy followed by slow-twitch muscle atrophy.

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.

Glucuronidation as a metabolic barrier against zearalenone in rat everted intestine.

Zearalenone (ZON), produced by Fusarium fungi, exhibits estrogenic activity. Livestock can be exposed to ZON orally through contaminating feeds such as cereals, leading to reproductive disorders such as infertility and miscarriage via endocrine system disruption. However, the details of ZON metabolism remain unclear, and the mechanism of its toxicity has not been fully elucidated. In this study, we investigated the kinetics of ZON absorption and metabolism in rat segmented everted intestines. ZON absorption was confirmed in each intestine segment 60 min after application to the mucosal buffer at 10 µM. Approximately half of the absorbed ZON was metabolized to α-zearalenol, which tended to be mainly glucuronidated in intestinal cells. In the proximal intestine, most of the glucuronide metabolized by intestinal cells was excreted to the mucosal side, suggesting that the intestine plays an important role as a first drug metabolism barrier for ZON. However, in the distal intestine, ZON metabolites tended to be transported to the serosal side. Glucuronide transported to the serosal side could be carried via the systemic circulation to the local tissues, where it could be reactivated by deconjugation. These results are important with regard to the mechanism of endocrine disruption caused by ZON.

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.

Analysis of Corticosterone and Testosterone Synthesis in Rat Salivary Gland Homogenates.

Extra-adrenal steroid hormone production has been reported in several tissues, the biological role of which is interesting in terms of hormonal regulation of metabolism, growth, and behavior. In this report, we describe for the first time steroidogenesis in rat salivary glands. Enzyme activities associated with corticosterone and testosterone production were detected in rat salivary glands by LC-MS analysis. In tissue homogenates of rat salivary glands, progesterone was produced enzymatically in vitro from pregnenolone in the presence of NADPH and NADH. Deoxycorticosterone was produced from progesterone, corticosterone from deoxycorticosterone, and testosterone from androstenedione (but not pregnenolone from cholesterol) via enzymatic reactions using the same tissue homogenates. Immunoblotting analysis indicated the expression of 11ß-hydroxylase (cytochrome P450 11ß1; CYP11ß1), which mediated the production of corticosterone from deoxycorticosterone. However, CYP family 11 subfamily A member 1 (CYP11A1)-mediated production of pregnenolone from cholesterol was not detected in the salivary glands by immunoblotting using a specific antibody. These results indicate that corticosterone and testosterone are produced from pregnenolone in rat salivary glands. The initial substrate in salivary steroidogenesis and the roles of salivary corticosterone and testosterone are discussed.

Rapid prolactin induction in adult male rats after treatment with diethylstilbestrol.

Diethylstilbestrol (DES) is a synthetic oestrogen known to disrupt the endocrine system and to cause reproductive toxicity mediated via the hypothalamic-pituitary-adrenal axis; however, its molecular mechanism of action is poorly understood. In the present study, we found that, after only 1 week of exposure to DES, blood testosterone dramatically decreased and that this decrease was associated with a strong induction of prolactin (PRL). Even with the increase in PRL, the luteinising hormone and follicle-stimulating hormone mRNAs slightly decreased. Our results show that, after 48 hours of a single dose of DES, there was a six-fold increase in PRL expression. After exploring the upstream mechanisms, we determined that dopamine, which inhibits PRL secretion in male rats, did not decrease in the pituitary gland of DES-treated rats, whereas vasoactive intestinal peptide (VIP), which mediates the acute release of PRL, was elevated. Serotonin (5-HT) increased in the brain of male rats 24 hours after a single DES treatment; however, PRL, VIP or 5-HT was not induced by DES in female rats. Our results indicate that DES induces the expression of pituitary PRL in male rats by stimulating VIP in the hypothalamus and 5-HT in the central nervous system.

Corticosterone biosynthesis in mouse clonal myoblastic C2C12 cells.

Corticosterone (CORT), the major glucocorticoid in rodents, is secreted from the adrenal gland, affects various organs in the body and regulates energy metabolism as a stress response. Although local steroidogenesis of androgens and estrogens in skeletal muscles has been previously reported, local CORT synthesis in skeletal muscle remains unconfirmed. In the present study, we investigated steroidogenic activities in a clonal myoblastic cell line, C2C12 cells. Three enzymes involved in CORT synthesis, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450c21 and cytochrome P45011ß, were identified in C2C12 cells by detecting the enzymatic reaction products with LC-MS/MS analysis. Only one enzyme that mediates cholesterol cleavage was not detected in the cells. After the addition of pregnenolone-sulfate conjugates to the cell culture medium, pregnenolone was detected and increased according to the incubation time. In conclusion, CORT synthesis occurs in C2C12 cells, and it is suggested that the initial steroidogenesis substrate is the pregnenolone-sulfate conjugate.