| Intrauterine growth restriction (IUGR) is a common birth defect that is often considered as an essential cause for a series of perinatal diseases including fetal distress, neonatal asphyxia and perinatal death. Besides, the adverse effects of IUGR can be prolonged to adulthood and result in physical and mental stunting as well as increased susceptibility to adult metabolic syndrome. Ethanol is one of the most widely consumed substances of abuse. Prenatal ethanol exposure (PEE) may induce a variety of adverse fetal outcomes, including morphological abnormalities and fetal growth retardation. The increasing evidences suggest that programmed alteration of the hypothalamic-pituitary-adrenal (HPA) axis is most likely to be involved in the mechanism underlying the IUGR-originated adult metabolic syndrome. Adrenal is the terminal organ of HPA axis that is responsible for the steroidogenesis, which is of great significance for maintaining normal gestation, promoting fetal growth and nervous system maturation. Low birth weight induced dysfunction of the adrenal glucocorticoid (GC) synthesis increases the risk of postnatal metabolic syndrome. Therefore, the programmed alteration of adrenal steroidogenesis may play an important role in IUGR originated adult metabolic syndrome. We have previously demonstrated that PEE resulted in fetal over-exposure to maternal GC and inhibition of the key/rate-limiting enzyme-steroidogenic acute regulation protein (StAR) and cytochrome P450 cholesterol side chain cleavage (P450scc) of fetal adrenal steroidogenesis. It suggested that PEE inhibit fetal adrenal steroidogenesis.Insulin-like growth factor 1 (IGF1) is a central factor in endocrine regulatory system, and modulates a variety of physiological activities in various tissues, including stimulation of pre-and post-natal cell proliferation, differentiation and metabolism. As we all know, IGF1 plays a role in fetal development. Recent study showed that constitutive ablation of IGF1R led to adrenal growth retardation and dysfunction in mice. It has been observed that IGF-1 and its receptor could modulate the expression of Steroidogenic factor-1 (SF-1) as well as relevant steroidogenic enzyme system and enhance the synthesis of steroid by phosphorylating PI3K/Akt signaling pathway. All of the above suggest that IGF-1 play vital role in modulating the adrenal corticosterone synthesis and secretion. GC acting on corticoid receptors (CR) in target tissues is determined not only by circulating GC levels, but also by intracellular GC metabolism regulated by two 11?-hydroxysteroid dehydrogenases (11?-HSDs) isoforms: 11?-HSD1 and 11?-HSD2. CR comprises the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) that are both ligand-dependent nuclear transcriptional factors. CCAAT/enhancer-binding proteins (C/EBPs) family, in which isoforms of C/EBP? and C/EBP? each act as essential collaborators in CR-mediated expression of downstream gene. C/EBPs can coordinate with CR to regulate downstream multiple gene expression (such as IGF1), in which activated C/EBP? inhibit gene expression of IGF1R while C/EBP?-deficient mice could severely reduce circulating IGF1 levels. It suggested that GC-metabolic activation system (11?-HSDs/CR/C/EBPs) might get involved in regulating IGF1 signaling pathway.Autophagy is the cellular catabolic process in which intracellular proteins/organelles are sequestered and then transported to and degraded in lysosomes to recycle cellular components under stress conditions or to eliminate damaged proteins/organelles. Autopahgy play a vital role in cell differentiation and embryonic development, and almost all tissues of newborn rats can be detected autophagy activation. The inhibition of autophagy in testicular leyding cells could reduce its testosterone synthesis. Ethanol could increase the level of the autophagic marker in SH-SY5Y neuroblastoma cells. It indicated that autophagy might be involved in modulating the adrenal steroidogenesis.Here, we established a rat IUGR model by PEE, and evaluated the adrenal developmental abnormalities of offspring. Furthermore, we explored its intrauterine programming mechanisms by investigating the serum phenotypes, gene expression of adrenal GC-metabolic activation system, IGF1 signaling pathway, and autophagy as well as steroidogenesis before and after birth. This work will be beneficial to better reveal the short-and long-term outcomes of ethanol developmental toxicity, and to clarify the susceptibility to metabolic syndrome in adult male offspring and its underlying mechanisms.PART ONEPrenatal ethanol exposure-induced adrenal developmental abnormality and IGF1-autophagy mechanism of fetal ratsObjective:To investigate the effects of prenatal ethanol exposure on the serum phenotypes, gene expression of adrenal GC-metabolic activation system, IGF1 signaling pathway, autophagy and steroidogenesis in vivo, and to address the regulatory mechanism.Methods:From GD11 to GD20, the pregnant rats were administered 4.0 mg/kg of ethanol per day to establish a stable IUGR rat model. The control group received the same volume of the vehicle. The adrenal histopathological and cytopathological examination was based on hematoxylin-eosin staining (H&E), and transmission electron microscope (TEM); immunohistochemistry was employed to detect the Ki67, Beclin 1 and LC3 expression of fetal adrenal; enzyme-linked immune sorbent assay (ELISA) was used to detect the level of blood IGF-1 and corticosterone (CORT), as well as adrenal CORT; real-time quantitative PCR was performed to measure mRNA expression of fetal adrenal genes, including SF-1, StAR, P450scc,3?-hydroxysteroid dehydrogenase (3?-HSD), steroid 21-hydroxylase (P450c21), steroid 11?-hydroxylase (P450c11), IGF-1 signaling pathway (IGF-1, IGF-1R and AKT1).Results: ?Body weight and IUGR rate:When being compared with control, the body weights were decreased of that of control (P<0.01); the male and female fetal IUGR rate reached to 79.7% and 85.4%(P<0.01), respectively. ? Pathological changes and cell proliferation:the male and female fetus adrenal cortex pathological changes, presenting cytoplasmic swelling deformation were observed in the PEE group. The entire cross-sectional area of fetal adrenal deceased in the PEE group (P<0.05, P<0.01). Immunohistochemical result showed that the number of Ki67-stained nuclei (red arrows) was reduced in fetal adrenal cortical cells compared with the control (P<0.05, P<0.01). Ultra-structural observation revealed that, in adrenal cortex cells from the PEE group, the mitochondrial structure appears to have fewer cross sections of the cristae. ? Adrenal steroidgenetic enzyme mRNA expression and CORT production:When being compared with control, PEE treatment significantly decreased the mRNA expression of male fetus adrenal StAR and 3?-HSD (P<0.01), as well as male fetus adrenal StAR, P450scc, P450c21 and P450c11 (P<0.05, P<0.01); both of male and female adrenal local CORT were decreased (P<0.05). ? Change of IGF1 signaling pathway and autophagy. when being compared with control, the level of male and fetal serum IGF1 concentration was decreased in the PEE group (P<0.05). The expression of key genes (IGF1, IGF1R, AKT1) of the IGF1 signaling pathway was a marked decreased relative to the control (P<0.05, P<0.01) in male. The expression of IGF1R and AKT1 was decreased (P<0.05, P<0.01), while IGF1 were increased (P<0.01) in females. Immunohistochemical result showed that the expression of Beclin 1 and LC3? were increased in both male and female fetuses adrenal. ?Serum CORT level and GC-metabolic activation system:The level of fetal serum CORT concentration was an increasing trend (P=0.07), and female fetal serum CORT concentration was increased (P<0.05). The levels of 11?-HSD1 expression and 11?-HSD1/11?-HSD2 expression ratio were increased, and GR, C/EBPa expression as well as C/EBPa/C/EBP? expression ratio were significantly increased, but C/EBP? expression in the PEE group was significantly decreased (P<0.05, P<0.01). In female, the levels of 11?-HSD1 expression and 11?-HSD1/11?-HSD2 expression ratio were decreased, while MR, GR, C/EBP? and C/EBP?/C/EBP? expression ratio were all significantly increased (P<0.05,P<0.01).Conclusion:Prenatal ethanol exposure could inhibit fetal adrenal corticosterone synthesis, but active adrenal autopahgy. This activation status of GC-metabolic system (11?-HSDs/CR/C/EBPs) might be involved in mediating circulating high GC to inhibit adrenal IGF 1-autophagy signaling pathway, and result in abnormal cell proliferation and functional differentiation in fetal adrenal.PART TWOEthanol exposure induced autopahgy and its potential biological significance in human fetal adrenal cellsObjective:To investigate the effects of autophagy on steroidogenic function in fetal adrenal NCI-H295A cells in vitro, and to elucidate the potential biological significance of autophagy during fetal adrenal cell development.Methods:The human fetal adrenal cortex cells were treated with ethanol with different concentrations (0,15,30,60 and 120 mM) for 8 h, or with 60 mM of ethanol for different times (1,3,5 days). Rapamycin (200 nM) and Bafilomycin Al (10 nM) were also used to evaluate the change of ethanol-induced autophagic flux. Cell viability was evaluated by MTS assay; the levels of cortisol in the media were measured by ELISA. The adrenal cytopathological examination was based on TEM; Western blotting was used to detect protein expression of LC3-?/LC3-?, P62 and p-mTOR.Results: ?Cell viability:Ethanol (0,15,30,60 and 120 mM) treatment for 1 day had no significant effect on cell viability, while treatment for 3 d and 5 d had no significant inhibition on cell viability. ?Time-dependent relationship of autopahgy and cytopathological examination:Compared with control, LC3-II/LC3-I expression ratio was dose-dependently increased by 60 mM ethanol at 8,12 and 24 h, and the corresponding cortisol concentration in the media was decreased. Furthermore, ultra-structural observation revealed that ethanol induced the occurrence of autophagic vacuoles. ?Dose-dependent relationship of autopahgy: Compared with control, LC3-?/LC3-? expression ratio was increased, while the concentration of cortisol in the media was decreased in ethanol groups (60 and 120 mM) (P<0.01). ? Autopahgy flux:Compared with control, the exposure of ethanol, autophagy activator Rapamycin and ethanol+Rapamycin induced increases in LC3-?/LC3-? expression ratio, while P62 was decreased. Moreover, Bafilomycin Al could inhibit the ethanol-induced downregulation of P62. ?Mechanism of ethanol-induced autophagy:When being compared with control, the LC3-?/LC3-? expression ratio was increased in ethanol groups (60 and 120 mM), while the expression of P62 and p-mTOR (ser2448) was decreased. ? Autophagy directly regulated adrenal steroidogenesis:When being compared with control, although no change in viability of ethanol, Bafilomycin Al and Bafilomycin Al ethanol group, the concentration of cortisol in the media was decreased (P<0.01) in all three groups, respectively. Furthermore, Rapamycin could promote the expression of SF-1. ? Cortisol and ethanol treatment inducing autophagy:Compared with control group, the group of ethanol, cortisol and ethanol+cortisol could induce LC3-? (P<0.05, P<0.01).Conclusion:Ethanol could directly inhibit the IGF1R/PI3K/AKT1/p-mTOR (ser2448) signaling pathway and then upregulated autophagy which facilitate adrenal steroid hormone synthesis. Autopahgy is protective response to ethanol-induced inhibition of steroidogenic function.PART THREEPrenatal ethanol exposure-induced the change of adrenal structure and function of IUGR offspring fed with normal diet and its possible mechanismObjective:To observe changes of the serum phenotypes, adrenal steroidogenic enzyme system, IGF1-autophagy signaling pathway, GC-metabolic activation system in prenatal ethanol exposure-induced IUGR offspring fed by normal diet after birth, and to clarify the programmed alteration of adrenal corticosterone synthesis function in IUGR offspring induced by prenatal ethanol exposure.Methods:IUGR rat model was established by prenatal ethanol (4 g/kg·d) exposure. The pregnant rats were allowed to deliver spontaneously at term, and the offspring were fed by normal diet until PW24 and then sacrificed to collect blood and adrenal tissue. The weights of the offspring were recorded at different periods after birth. The adrenal histopathological and cytopathological examination was based on H&E, and immunohistochemistry was employed to detect the Ki67; ELISA assay was used to detect the level of blood IGF-1 and CORT, as well as adrenal CORT; Western blotting was use to measure the levels of LC3; Real-time PCR was to determine the mRNA expressions of steroidogenic enzymes (SF-1, StAR, P450scc, 3?-HSD, P450c21 and P450c11), GC-metabolic activation system and IGF-1 signaling pathway (IGF-1, IGF-1R and AKT1).Results:?Body weight and body weight gain rate:When being compared with control, the male offspring rats in the PEE group showed significantly lower body weights PW1 (P<0.01). After weaning (PW4), body weights of the PEE group showed no significant difference, but the corresponding gain rate showed a significant increase (P<0.01). The female offspring rats showed significantly lower body weights (P<0.05, P<0.01), and gain rate also showed a significant increase (P<0.01). ? Pathological changes and cell proliferation:The male and female offspring adrenal cortex pathological changes, disorganized cell arrangements with cytoplasmic swelling deformation in the offspring adult adrenal glands were observed in the PEE groups. The entire cross-sectional area of adult adrenal showed no obvious change. Compared with the control, the immunohistochemical result showed that the number of Ki67-stained nuclei (red arrows) was not significantly different in adult adrenal cortical cells. ?Adrenal steroidgenetic enzyme mRNA expression and CORT production:When being compared with control, PEE significantly decreased the mRNA expression of adrenal SF1 and StAR (P<0.05) but significantly increased 3(3-HSD and P450c21 in male adult adrenal (P<0.05). The mRNA expression of adrenal SF-1, StAR, 3?-HSD, P450c21 and P450c11 were all obviously decreased in female offspring rats (P<0.05, P<0.01). Moreover, ELISA results showed that the male and female adult offspring adrenal CORT content in the PEE group were 90.1% and 77.1% of that of the control group. ? Change of IGF1 signaling pathway and autophagy:when being compared with control, the male offspring rats showed an increasing trend of serum IGF1 level, and IGF1 level increased (P<0.05) in female offspring rats. The expression of key genes (IGF1, IGF1R, AKT1) of the IGF1 signaling pathway was a markedly increased in male adrenal relative to the control (P<0.05, P<0.01), while the IGF1 signaling pathway was no obvious change in female adrenal. Whatmore, the LC3-?/LC3-? expression ratio was no obvious change in both male and female adult offspring rats. ?Serum CORT level and GC-metabolic activation system: Compare with the control, the male offspring rats showed a decrease in serum CORT level (P<0.01), female serum CORT concentration was a decreasing trend (P=0.069). Furthermore, in male, the levels of 11?-HSD1 expression and 11?-HSD1/11?-HSD2 expression ratio were decreased (P<0.05, P<0.01), while C/EBP? expression was significantly increased (P<0.05). In female, the levels of C/EBP?, C/EBP?/C/EBP? expression ratio were all significantly increased (P<0.05, P<0.01).Conclusion:In the present study, for the first time we demonstrated that prenatal ethanol exposure induced adrenal developmental abnormality in offspring rats, which may have resulted from a "two-programming. The intrauterine lower functional programming of adrenal steroidogenesis is "the first programming", which results in fetal adrenal developmental abnormality. A GC-metabolic activation system-related GC-IGF1-autophagy system is "the second programming", which maintains a stable enviroment of adrenal tissue, and promote catch-up growth of postnatal adrenal structure and function. These findings will be beneficial in elucidating the fetal developmental toxicities of ethanol, and provide an underlying mechanisms for clarifying the susceptibility to HPA axis-associated diseases (e.g. metabolic illness) for IUGR offspring.PART FOURThe dysfunction of adrenal and insulin resistance in of ethanol-induced IUGR offspring fed by high-fat diet/chronic stress after birthObjective:To mimic the high-fat diet in human, and further observe the change of adrenal corticosterone synthesis function and glucose metabolism, as well as the mechanism of ethanol-induced IUGR offspring fed by high-fat diet/chronic stress after birth.Methods:IUGR rat model was established by prenatal ethanol (4 g/kg-d) exposure, the weights of the offspring were recorded at different periods after birth. The first batch of offspring were fed by high-fat diet (HFD) containing 89.5% of corn flour,10% of lard and 0.5% of cholesterol from the day after weaning. On PW24, oral glucose tolerance test (OGTT) was performed. The second batch of offspring were also fed by high-fat diet. At PW21, the rats were exposed to an unpredictable chronic stress (UCS) procedure for 21 days, and the stresses were administered once daily between 8:30 am and 10:30 am with lights-on, except the 24 h duration stressors. Stressors consisted:food deprivation for 24 h; water deprivation for 24 h; tail pinch for 5 min; hot stress in oven at 45? for 5 min; cold swimming at 4-8? for 4 min, after which they were towel dried; reversed day and night cycles; social isolation (one rat per cage) for 24 h. Every stressor was administered randomly at an interval of 7 days (three times within 21 days). On PW24, OGTT was performed. At the last day of stress after 12 h fasting, all rats were obliged to swim at 4-8? for 4 min and were towel dried. One hour after swimming, rats were anesthetized with isoflurane and decapitated to collect blood and adrenal samples. Biochemical methods and ELISA techniques were used to detect serum glucose, CORT and insulin concentrations, and Radioimmunoassay was employed to detect serum adrenocorticotropic hormone (ACTH). The adrenal histopathological examination was based on H&E. Real-time PCR was performed to determine the mRNA expressions of steroidogenic enzymes system (SF-1, StAR, P450scc,3?-HSD, P450c21 and P450c11), GC-metabolic activation system and IGF-1 signaling pathway (IGF-1, IGF-1R and AKT1).Results: ? Body weight:Compared with control, PEE resulted in decreased body weights of the PEE male and female pups at PW1 (P<0.01). After weaning, the body weights of male and female offspring rats in PEE group were close to that of control when fed with HFD (P<0.01, P<0.05), but the corresponding body weight growth rate were all significant higher related to control at each time point (P<0.01, P<0.05), respectively. ?Serum ACTH and CORT level:When being compared with control, ACTH and CORT level in male and female of ethanol group were decreased (P<0.01) or a decreasing trend before stress. While under UCS, the ACTH and CORT level of male and female in the ethanol group were increased (P<0.01, P<0.05) or an increasing trend. The corresponding gain rate of ACTH and CORT were all increased (P0<.01). ?Adrenal steroidogenic enzyme system:when being compared with high-fat diet, the mRNA expression of adrenal StAR, P450scc,3?-HSD, P450c11 were all decreased in male of ethanol group (P?0.05, P<0.01); for the female of ethanol group, the mRNA expression of P450c11 were decreased (P<0.05) while that of P450c21 was increased (P<0.05), the StAR, P450scc expression showed no alteration. After UCS, the mRNA expression of adrenal P450scc,3?-HSD, P450c11 were all increased in male of ethanol group (P<0.05, P<0.01); for the female of ethanol group, the mRNA expression of SF-1, StAR, P450scc and P450c11 were increased (P<0.05, P<0.01).? Change of IGF1-autophagy signaling pathway:when being compared with control, the expression of key genes (IGF1 and IGFIR) of the IGF1 signaling pathway was a markedly increased relative to the control (P<0.05) in male, and expression of IGF1, IGFIR and AKT1 were increased (P=0.09, P<0.05, P<0.01) in female. Moreover, the LC3-II/LC3-I expression ratio was no obvious change in both male and female adult offspring rats before stress. After UCS, the expression of IGF1 was a markedly decreased relative to the control (P<0.05) in male, and expression of IGF1 and IGFIR were decreased (P<0.01) in females. The corresponding LC3-?/LC3-? expression ratio was increased in both male and female adult offspring rats. ? Serum glucose, insulin and insulin resistance index (IRI):Compared with the control group, the basal level of serum glucose and IRI in male were significantly increased (P<0.05), and that of in the female was no obvious change. After UCS, serum glucose and IRI in male were significantly increased (P<0.05), while serum insulin and IRI were a decreasing trend. ? Glucose metabolism:when being compared with control, after glucose challenge, the relative serum glucose at 30 and 120 min as well as area under the curve (AUC) of PEE group were all increasing trend, and the insulin and AUC were all increased (P<0.05, P<0.01) in male. In female, relative serum glucose and AUC were not changed, while decline tendencies of insulin at 120 min and AUC were observed. After UCS, in male, the relative serum glucose at 30,60 and 120 min as well as AUC of PEE group were decreased or decreasing trend (P=0.06, P=0.08, P<0.05, P<0.01), while relative serum insulin at 30 min was increasing trend, and relative serum insulin at 60 and 120 min were an increasing trend. Meanwhile, in female, relative serum glucose and AUC were no change, while relative serum insulin was an increasing trend except for 60 min.Conclusion:Prenatal ethanol exposure induced adrenal developmental abnormality in offspring rats, which may have resulted from a "two-programming. The intrauterine lower functional programming of adrenal steroidogenesis is "the first programming", which results in fetal adrenal developmental abnormality, A GC-metabolic activation system-related GC-IGF1-autophagy system is "the second programming", which promote catch-up growth of postnatal adrenal function by high-fat diet, and maitain the high level of adrenal function by high-fat diet with unpredictable chronic stress. The enhanced stress sensitivity of adrenal might lead to insulin resistance and further induce diabetic-like changes for IUGR offspring.Summary:1. In the present study, PEE could inhibit fetal adrenal corticosterone synthesis, but active adrenal autopahgy. This activation status of GC-metabolic system might be involved in mediating circulating high GC and ethanol to inhibit adrenal IGF1-autophagy signaling pathway, and result in abnormal cell proliferation and functional differentiation in fetal adrenal.2. Ethanol could directly inhibit the IGF1R/PI3K/AKT1/p-mTOR (ser2448) signaling pathway and then upregulated autophagy which is protective response to ethanol-induced inhibition of steroidogenic function.3. PEE induced adrenal developmental abnormality in offspring rats, which may have resulted from a "two-programming". The intrauterine lower functional programming of adrenal steroidogenesis is "the first programming", which results in fetal adrenal developmental abnormality. A GC-metabolic activation system-related GC-IGF1-autophagy system is "the second programming", which maintains a stable enviroment of adrenal tissue, and promote the catch-up growth of postnatal adrenal structure and function with normal diet and HFD.4. After UCS, the GC-IGF1-autophagy system could maintain the high level of adrenal function by high-fat diet with unpredictable chronic stress. The enhanced stress sensitivity of adrenal might lead to insulin resistance and further induce diabetic-like changes for IUGR offspring. |
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