BmCncC/keap1-pathway is involved in high-temperature induced metamorphosis regulation of silkworm,Bombyx mori
Jinxin LiTingting MaoZhengting LuMengxue LiZhengting LuJianwei QuYilong FangJian ChenHui WangXiaoyu ChengHu JiahuanYu LiuZiyan ZhangGu ZhiyaLi FanchiBing Li
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Abstract The global warming has affected the growth, development and reproduction of insects. However, the molecular mechanism of high temperature stress-mediated metamorphosis regulation of lepidopteran insect has not been elucidated. In this study, the relationship between the insect developmental process and endogenous hormone level was investigated under high temperature (36 ° C) stress in Bombyx mori ( B. mori ). The results showed that the duration of 5 th instar larvae were shortened by 28 ± 2 h, and the content of 20E was up-regulated significantly after 72 h of high temperature treatment, while the transcription levels of 20E response genes E93, Br-C, USP, E75 were up-regulated 1.35, 1.25, 1.28, and 1.27-fold, respectively. The high temperature treatment promoted the phosphorylation level of Akt and the downstream BmCncC/keap1 pathway was activated, the transcription levels of 20E synthesis-related genes cyp302a1, cyp306a1, cyp314a1 and cyp315a1 were up-regulated by 1.12, 1.51, 2.17 and 1.23-fold, respectively. After treatment with double stranded RNA of BmCncC (dsBmCncC) in BmN cells, the transcription levels of cyp302a1 and cyp306a1 were significantly decreased, whereas up-regulated by 2.15 and 1.31-fold, respectively, after treatment with CncC activator Curcumin. These results suggested that BmCncC/keap1-mediated P450 genes ( cyp302a1, cyp306a1 ) expression resulted in the changes of endogenous hormone level, which played an important role in the regulation of metamorphosis under high temperature stress. Studies provide novel clues for understanding the CncC/keap1 pathway-mediated metamorphosis regulation mechanism in insects. Author Summary Mammalian nuclear transcription factor Nrf2 (NF-E2-related factor 2) plays an important role in the stress response of cells. CncC is a homolog of mammalian Nrf2 in insect, regulating the genes expression of insect antioxidant enzymes and cytochrome P450 detoxification enzyme. Evidence suggests that the CncC/Keap1 pathway also plays an important role in regulating insect development. Here, we investigated the regulatory mechanism between the CncC/Keap1 pathway and metabolism of silkworm hormones in Lepidoptera. We found that high temperature induction accelerated the development of silkworm, the ecdysone content and related metabolic genes in hemolymph were significantly up-regulated, the CncC/Keap1 pathway was activated, and the expression of BmCncC was significantly increased, indicating that the Cncc/Keap1 pathway plays an important role in this process. The expression of cyp302a1 and cyp306a1 was significantly decreased by RNA interference with BmCncC , which indicated that CncC in silkworm had a regulatory relationship with downstream 20E synthetic gene. In summary, the results indicate that the CncC/Keap1 pathway plays an important role in regulating hormone metabolism in silkworm, providing a basis for further study of the relationship between CncC/Keap1 pathway and development in insects.Keywords:
KEAP1
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Abstract The global warming has affected the growth, development and reproduction of insects. However, the molecular mechanism of high temperature stress-mediated metamorphosis regulation of lepidopteran insect has not been elucidated. In this study, the relationship between the insect developmental process and endogenous hormone level was investigated under high temperature (36 ° C) stress in Bombyx mori ( B. mori ). The results showed that the duration of 5 th instar larvae were shortened by 28 ± 2 h, and the content of 20E was up-regulated significantly after 72 h of high temperature treatment, while the transcription levels of 20E response genes E93, Br-C, USP, E75 were up-regulated 1.35, 1.25, 1.28, and 1.27-fold, respectively. The high temperature treatment promoted the phosphorylation level of Akt and the downstream BmCncC/keap1 pathway was activated, the transcription levels of 20E synthesis-related genes cyp302a1, cyp306a1, cyp314a1 and cyp315a1 were up-regulated by 1.12, 1.51, 2.17 and 1.23-fold, respectively. After treatment with double stranded RNA of BmCncC (dsBmCncC) in BmN cells, the transcription levels of cyp302a1 and cyp306a1 were significantly decreased, whereas up-regulated by 2.15 and 1.31-fold, respectively, after treatment with CncC activator Curcumin. These results suggested that BmCncC/keap1-mediated P450 genes ( cyp302a1, cyp306a1 ) expression resulted in the changes of endogenous hormone level, which played an important role in the regulation of metamorphosis under high temperature stress. Studies provide novel clues for understanding the CncC/keap1 pathway-mediated metamorphosis regulation mechanism in insects. Author Summary Mammalian nuclear transcription factor Nrf2 (NF-E2-related factor 2) plays an important role in the stress response of cells. CncC is a homolog of mammalian Nrf2 in insect, regulating the genes expression of insect antioxidant enzymes and cytochrome P450 detoxification enzyme. Evidence suggests that the CncC/Keap1 pathway also plays an important role in regulating insect development. Here, we investigated the regulatory mechanism between the CncC/Keap1 pathway and metabolism of silkworm hormones in Lepidoptera. We found that high temperature induction accelerated the development of silkworm, the ecdysone content and related metabolic genes in hemolymph were significantly up-regulated, the CncC/Keap1 pathway was activated, and the expression of BmCncC was significantly increased, indicating that the Cncc/Keap1 pathway plays an important role in this process. The expression of cyp302a1 and cyp306a1 was significantly decreased by RNA interference with BmCncC , which indicated that CncC in silkworm had a regulatory relationship with downstream 20E synthetic gene. In summary, the results indicate that the CncC/Keap1 pathway plays an important role in regulating hormone metabolism in silkworm, providing a basis for further study of the relationship between CncC/Keap1 pathway and development in insects.
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The physiological and pharmacological functions of the 20-kDa human GH (20K-hGH) isoform are unknown. We conducted a pharmacokinetic study of recombinant 20K-hGH in human subjects (Phase I clinical trial). Placebo or 20K-hGH was administered sc to normal men (20–31 yr of age, n= 6–8 per group) at 2100 h. Serum 20K- and 22K-hGH levels were monitored every 30 min for 24 h by specific enzyme-linked immunosorbent assays. Serum free fatty acid, insulin-like growth factor I, insulin, and glucose levels were measured for 24 h. In the placebo group, the secretion profiles of endogenous 20K- and 22K-hGH were pulsatile and similar to each other. The proportion of 20K- to 22K-hGH was fairly constant. In the 20K-hGH-treated groups, serum 20K-hGH levels increased in a dose-dependent manner over the dose range of 0.01–0.1 mg/kg. Maximum serum 20K-hGH levels were reached at 3–4 h and decreased with half-lives of 2–3 h. Marked suppression of endogenous 22K-hGH secretion was observed in a time-dependent manner. Serum free fatty acid and insulin-like growth factor I levels were significantly elevated (P < 0.01) at 4, 8, and 12 h and at 8, 12, and 24 h after 20K-hGH administration, respectively. Serum insulin and glucose levels did not change significantly within 24 h. These results suggested that: 1) regulation of 20K-hGH secretion is physiologically the same as that of 22K-hGH; 2) the pharmacokinetics after sc injection of 20K-hGH are comparable with those of 22K-hGH; 3) 20K-hGH regulates hGH secretion through "GH-induced negative feedback mechanisms"; and 4) administration of 20K-hGH is expected to exert GH actions (growth-promoting activity and lipolytic activity). Monitoring of serum 20K- and 22K-hGH levels may be useful in evaluating the effects of administered GH isoforms on their own release from the pituitary.
Endogenous growth theory
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Adrenal steroids have been shown to accelerate both spontaneous and thyroid hormone (TH)-induced metamorphosis. The present study is concerned with the mechanisms underlying this effect. Premetamorphic Rana catesbeiana tadpoles were immersed in water containing 1–20 nM T4 or T3 ± 1 fiM corticosterone (B) for 6 to 19 days. B is the predominant glucocorticoid in tadpole plasma before climax. As indicated by the rate of tail resorption and hepatic carbamyl phosphate synthetase (CPS) activity, metamorphosis was significantly accelerated when TH-treated animals also received B: after 14 days in T4 (20 nM), tadpole tail length was decreased by 10%; in T4 + B, the decrease was 19%. At the same time, CPS activity was increased approximately 12-fold in tadpoles in T4 and 18- fold in those in T4 + B. Comparable results were obtained when T3 was employed. Neither tail resorption nor CPS activity was influenced by B alone. Plasma T4 levels attained at all bath concentrations of T4 were increased more than 2-fold when B was present. The same was true when T3 was used. In addition, in tadpoles immersed in 20 nM T4, the plasma T3 level was significantly increased in the presence of B. Assessment of 5'- deiodinase and 5-deiodinase activities in vitro revealed that administration of B for 7 days resulted in a significant increase in 5'-deiodinase activity in skin and a significant reduction in T3 5-deiodinase activity in both liver and gut. Treatment with B reduced the rate of turnover of T3; 72 h after injection of 0.01 nmol [125I]T3, B-treated tadpoles had retained approximately 58% of the dose vs. 42% in controls. T3 nuclear receptor number (sites per nucleus) as assessed by an in vitro saturation technique was not altered in liver, tail, or red blood cells after 14 days of exposure to B. On the basis of these findings it is suggested that the acceleration of TH-induced metamorphosis by B is due at least in part to its ability to increase the plasma level of- T4 or T3 through its effects on the peripheral metabolism of TH. (Endocrinology127: 2997–3002,1990)
Tadpole (physics)
Corticosterone
Iodothyronine deiodinase
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In the adult male Wistar rat a 2-fold 2-min restraint stress exposure, repeated 15 min apart, activated the adrenocortical secretion more than a single one would have. However, in rats with a pharmacological block of the endogenous CRF release, exogenous CRH (0.3 micrograms/kg iv), administered 15 min after a first similar dose, was unable to stimulate pituitary-adrenocortical activity above the level attained with the first peptide injection. On the contrary, in the same conditions exogenous arginine vasopressin (AVP) (0.3 micrograms/kg iv) administered 15 min after CRH, was able to further stimulate pituitary-adrenocortical activity. Using the same experimental procedure, oxytocin (0.3 micrograms/kg iv) was found to be totally inactive. The physiological import of these findings was investigated in the Brattleboro rat, genetically lacking in endogenous AVP, in which, unlike the control Long-Evans strain, the 2-fold stress exposure did not cause an increase in plasma corticosterone concentration greater than that of a single exposure. These results suggest that endogenous AVP is essential in sustaining adrenocortical activation in circumstances in which pituitary refractoriness towards CRH stimulation intervenes.
Corticosterone
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The possible physiological significance of endogenous inhibin has been evaluated in the female rat during sexual maturation. Plasma inhibin levels measured by a specific RIA were very low, but detectable at 5 days of age, then progressively increased until day 17, when they showed an abrupt rise. This rise was concurrent with a dramatic fall in plasma FSH levels. At day 30, inhibin values were not significantly (P greater than 0.05) different from those of adult females. The immunoneutralization of endogenous inhibin had no statistically significant (P greater than 0.05) effect on plasma FSH levels in 10-day-old immature female rats, but markedly (P less than 0.01) increased them at day 20 and 30. These results suggest that endogenous inhibin plays a physiological role in modulating FSH secretion from day 20 of age in the female rat.
Sexual maturity
Plasma levels
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Iodothyronine deiodinase
Ambystoma mexicanum
Corticosterone
Hypothalamic–pituitary–thyroid axis
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Diurnal changes in plasma corticosterone concentrations of rats are supposed to be controlled by low amplitude changes in plasma adrenocorticotropin (ACTH) that run in phase with marked changes in adrenal sensitivity to ACTH (1, 2). These sensitivity changes are probably under neuronal control (3), but the mechanisms remain to be elucidated. By the use of a highly specific monoclonal antibody to rat corticotropin‐releasing factor (CRF) (4), we studied the role of endogenous CR‐ in these processes. Blockade of endogenous CRF prevented the evening rise in plasma ACTH and reduced corticosterone levels to less than 10%. The CRF antibody did not affect morning ACTH concentrations but nevertheless reduced morning corticosterone levels by 40% to 60%. In dexamethasone‐treated rats, immunoneutralization of endogenous CRF caused a similar reduction in the plasma corticosterone response to exogenous ACTH. We conclude that endogenous CRF plays a physiological role in controlling the adrenal sensitivity to ACTH.
Corticosterone
Corticotropin-releasing hormone
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The plasma ACTH response to corticotrophin releasing factor (CRF) was studied in seven normal individuals. Five subjects were restudied following 4 d of salt restriction which resulted in raised endogenous plasma angiotensin II secretion. In a third experiment six subjects were given CRF following pre-infusion of hypertonic saline which significantly increased endogenous plasma vasopressin (AVP) levels. We were unable to demonstrate that high endogenous plasma AII levels were associated with a significant change in the plasma ACTH or cortisol responses to CRF. However there was an almost three-fold increase in the ACTH response when endogenous plasma AVP was elevated by hypertonic saline. It is concluded that AVP is likely to be of physiological importance in potentiating the ACTH response to CRF and that AVP and CRF together may provide a better test of pituitary ACTH secretion than either peptide alone.
Hypertonic saline
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The present studies examined the effects of streptozotocin-induced diabetes on the dynamics of GH, insulin (IRI), and glucose secretion in 68 chronically cannulated male rats. Animals were rendered diabetic by a single iv injection of streptozotocin (65 mg/kg) and sampled every 15 min for periods of 6h both before and at various times after treatment. The typical ultradian rhythm of GH secretion was evident in normal rats, with most peak GH values greater than 500 ng/ml (mean 6-h GH level, 125.3 ± 17.2 ng/ml). After streptozotocin administration, a significant depression in the amplitude of GH pulses was observed as early as 18 h post treatment (mean 6-h GH level, 54.4 ± 9.4 ng/ml; P < 0.01) concomitant with a marked elevation in plasma glucose levels and severe depression of plasma IRI levels. The amplitude and duration of the GH secretory episodes continued to decline over a 4-week period in the presence of persisting hyperglycemia and mostly undetectable plasma IRI levels. The pituitary GH concentration was not significantly altered 8 days post treatment. To elucidate the mechanism mediating the streptozotocininduced GH suppression response, the role of somatostatin (SRIF) was assessed by passive immunization with a specific SRIF antiserum. Two groups of streptozotocin-diabetic rats were administered iv 1 ml of either SRIF antiserum or normal sheep serum. The GH profiles of diabetic rats administered normal sheep serum continued to show a marked suppression of GH pulses (mean 6-h GH level, 69.5 ±7.8 ng/ml). In striking contrast, the GH profiles of diabetic SRIF antiserum-treated rats revealed a rapid (within 15–30 min) restoration of high amplitude GH pulses (500–800 ng/ml) and a significant elevation of GH trough levels. The mean 6-h GH level in these animals (150.3 ± 16.8 ng/ml) was significantly greater than that in normal sheep serumtreated controls (P < 0.01). In contrast to the effects on GH, no significant differences were observed in plasma glucose and IRI levels of diabetic SRIF antiserum-treated rats compared to diabetic normal sheep serum-treated controls. These results clearly demonstrate that the GH secretory episodes are markedly suppressed in the presence of streptozotocin-induced diabetes. Furthermore, the findings indicate that circulating SRIF plays a role in the GH suppression response observed in streptozotocin diabetes but is not involved in mediating the streptozotocin-induced effects on glucose and IRI.
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The effect of acute activation of the ACTH-adrenal axis on circulating testosterone (T) levels was investigated. Elevation of circulating cortisol resulting from insulin-induced hypoglycemia or the administration of hydrocortisone was followed by a rapid decrease in serum T levels, without accompanying changes in LH or PRL. These findings suggest that hypercortisolism of endogenous or exogenous sources suppresses T secretion by a direct action on the testis. This adrenal-testicular axis may have biological implications on the reproductive adaptation to stress.
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