Pituitary GH-secretory profiles are sex dependent and regulate the sexually dimorphic expression of a large number of genes in the liver. The slow response of many sex-specific liver genes to changes ...in plasma GH status suggests that GH acts in the liver via both direct and indirect mechanisms organized in a hierarchical regulatory network. Presently, genome-wide liver transcription profiling was conducted to elucidate the global impact of pituitary hormone ablation on the sex specificity of rat liver gene expression and to identify sex-specific genes that respond rapidly to GH as candidates for direct targets of GH action. Hypophysectomy abolished the sex specificity of approximately 90% of 1032 sex-dependent genes, consistent with the dominant role of pituitary GH in regulating liver sexual dimorphism. Two major classes of sex-specific genes were identified: genes that were down-regulated after hypophysectomy and may be subject to positive GH regulation (461 class I genes), and genes that were up-regulated after hypophysectomy and may be subject to negative GH regulation (224 class II genes). Fifty class I sex-specific genes were induced, and 38 class II sex-specific genes were suppressed within 90 min of a physiological GH pulse, suggesting they are primary GH response genes. A further 71 sex-specific genes responded after a second GH treatment and may correspond to secondary response genes. Twenty four DNA-binding proteins were identified as early GH response genes, of which 15 were induced and nine were suppressed by GH. Five of these 24 genes displayed sex-specific expression, consistent with a hierarchical transcriptional network controlling sex-specific liver gene expression. Class II male-specific genes, such as Cyp2a2 and Cyp2c13, were down-regulated within 30 min of GH pulse treatment, as determined by heterogeneous nuclear RNA analysis, suggesting that transcription of these genes is restricted to the GH-free interpulse period in adult male rat liver. We conclude that GH acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression.
Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a ...global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class I) and genes subject to negative regulation by pituitary hormones (class II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30–90 min of GH pulse treatment at a physiological dose were identified as putative direct targets of GH action (early response genes). Intrinsic sex differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were induced by GH within 30 min in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor myocyte enhancer factor 2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex differences in predisposition to liver cancer or other hepatic pathophysiologies.
Intrinsic sex-differences characterize liver GH responsiveness, with 29 male-specific targets of the transcription factor Mef2 induced rapidly in male but not female hypophysectomized mouse liver.
Structural and functional abnormalities in the tumor vascular network are considered factors of resistance of solid tumors to cytotoxic treatments. To increase the efficacy of anticancer treatments, ...efforts must be made to find new strategies for transiently opening the tumor vascular bed to alleviate tumor hypoxia (source of resistance to radiotherapy) and improve the delivery of chemotherapeutic agents. We hypothesized that Botulinum neurotoxin type A (BoNT-A) could interfere with neurotransmitter release at the perivascular sympathetic varicosities, leading to inhibition of the neurogenic contractions of tumor vessels and therefore improving tumor perfusion and oxygenation.
To test this hypothesis, BoNT-A was injected locally into mouse tumors (fibrosarcoma FSaII, hepatocarcinoma transplantable liver tumor), and electron paramagnetic resonance oximetry was used to monitor pO(2) in vivo repeatedly for 4 days. Additionally, contrast-enhanced magnetic resonance imaging was used to measure tumor perfusion in vivo. Finally, isolated arteries were mounted in wire myograph to monitor specifically the neurogenic tone developed by arterioles that were co-opted by the surrounding growing tumor cells.
Using these tumor models, we showed that local administration of BoNT-A (two sites; dose, 29 units/kg) substantially increases tumor oxygenation and perfusion, leading to a substantial improvement in the tumor response to radiotherapy (20 Gy of 250-kV radiation) and chemotherapy (cyclophosphamide, 50 mg/kg). This observed therapeutic gain results from an opening of the tumor vascular bed by BoNT-A because we showed that BoNT-A could inhibit neurogenic tone in the tumor vasculature.
The opening of the vascular bed induced by BoNT-A offers a way to significantly increase the response of tumors to radiotherapy and chemotherapy.
Ontogenic changes in the rat bile acid (BA) pool, measured enzymatically and by GC-MS, and expression of enzymes (5α-reductase, 5β-reductase, and cytochrome P450 enzymes Cyp7a1, Cyp8b1, Cyp27 and ...Cyp3a11), transporters bile salt export pump, sodium taurocholate-cotransporting polypeptide, apical sodium-dependent bile acid transporter, and organic solute transporter α/β (Ostα/Ostβ), and nuclear receptors fetoprotein transcription factor (Ftf), farnesoid X receptor (Fxr), small heterodimer partner (Shp), and hepatic nuclear factor 4α (HNF-4α), determined by quantitative PCR, were investigated. The absolute size of the BA pool increased progressively up to adulthood, whereas the complexity of its composition was high in fetuses, decreased after birth, increased again progressively up to adulthood, and decreased in aged animals. Allo-cholic acid only appeared early in development, in spite of low 5α-reductase expression. The relative size of the BA pool, corrected by liver weight, was maintained from 1 week after birth, except at weaning, when a transient peak accompanied by Shp downregulation and Cyp7a1 upregulation was observed. An imposed weaning delay of 1 week had no effect on the time course of the BA pool size but decreased the proportion of chenodeoxycholic and α-muricholic acids, whereas the proportion of cholic acid was increased, probably as a result of Cyp8b1 upregulation. In conclusion, changes in the expression of genes involved in BA homeostasis may play a role in physiological adaptations to digestive functions during the rat life span.
The effect of ageing on CYP3A2, a male specific isoform, was examined in adult (9 months) and senescent (24 months) male rats. A significant decrease (65%) of CYP3A2-related activity (midazolam ...oxidation) was observed in all senescent rats. Half of these rats still express CYP3A2 suggesting that decreased activities in these rats are due to post-translational modifications. The other senescent male rats did not express CYP3A2 anymore, indicating an impairment of transcription. These transcriptional modifications are due to the previously shown continuous secretion of GH in senescent male rats. GH also regulates HNF4α, a hepatocyte nuclear factor, essential for the basal transcriptional activation of the CYP3A2 gene. In senescent rats, a drastic reduction (76%) of HNF4α protein content and a decrease in DNA binding activity were observed. When these parameters were assessed in male and female rats of the same age (3 months), a higher HNF4α DNA binding activity and a higher HNF4α protein content (38%) were observed in female rats. Our results show that in male senescent rats (1) the decrease of HNF4α is not consistent with the continuous secretion of GH, and (2) the suppression of CYP3A2 expression is not dependent to the HNF4α binding activity.
The effect of ageing on CYP2E1 activity and its protein and mRNA contents was investigated in both adult (9 months) and senescent (24 months) male Wistar rats. The CYP2E1 activity (as measured by ...chlorzoxazone hydroxylation) was significantly decreased by 36% in senescent rats as compared to adult rats. However, this decrease of activity was not associated with a loss of protein content because the amount of both CYP2E1 protein and CYP2E1 mRNA did not decrease in senescent rats but rather increased, by 79% and 64% respectively, as compared to adult rats. Lipid peroxidation was increased significantly by 140% with ageing. The decrease in CYP2E1 activity could be explained by post-translational modification of CYP2E1 proteins, due to an increase in oxidative stress in senescent animals, leading to a loss of their functionality. However, no changes in the extent of protein carbonyls were observed in the adult versus senescent rats (16.2
±
9.6 vs. 12.7
±
7.3 nmol/mg prot) and the major proteasome activity remained unchanged. With regards to the increase of CYP2E1 expression, our results showed that the amount of hepatocyte nuclear factor 1α mRNA, a transcription factor that positively regulates CYP2E1, was strongly increased (154%) in senescent rats.
Ageing affects drugs metabolism influencing the therapeutic efficacy and safety of drugs. By using the experimental model of aged male rats, we investigated the influence of ageing on some CYP2C ...isoforms, the most important CYP450 sub-family in rats. The activity of the male specific CYP2C11 is decreased by 55% in senescent male rats. This correlates with a significant reduction of both protein content (80%) and mRNA (60%) indicating a demasculinization process. The expression of CYP2C12, a female specific isoform, is induced in senescent male rats indicating a feminization process. Neither the activity nor the expression of CYP2C6, a female predominant isoform, is modified in senescent male rats. Thereafter, certain putative GH mediators like some liver enriched transcription factors (LETFs) or STAT5b were investigated. The amount of HNF3β mRNA, a transcription factor involved in the up-regulation of CYP2C12, has been shown to increase by about three-fold in senescent male rats. With regard to STAT5b, which has been reported to be involved in the male specific regulation of CYP2C11, large amounts of phosphorylated STAT5 were observed in the liver of senescent male rats. These results indicate that while the induction of CYP2C12 during ageing could be due, at least partially, to the enhanced HNF3β expression, the decline of CYP2C11 is unlikely related to a decrease of STAT5 activation.
Sexual dimorphism in mammalian liver impacts genes affecting hepatic physiology, including inflammatory responses, diseased states, and the metabolism of steroids and foreign compounds. Liver sex ...specificity is dictated by sex differences in pituitary growth hormone (GH) secretion, with the transcription factor signal transducer and activator of transcription (STAT)5b required for intracellular signaling initiated by the pulsatile male plasma GH profile. STAT5a, a minor liver STAT5 form >90% identical to STAT5b, also responds to sexually dimorphic plasma GH stimulation but is unable to compensate for the loss of STAT5b and the associated loss of sex-specific liver gene expression. A large-scale gene expression study was conducted using 23,574-feature oligonucleotide microarrays and livers of male and female mice, both wild-type and Stat5a-inactivated mice, to elucidate any dependence of liver gene expression on STAT5a. Significant sex differences in expression were found for 2,482 mouse genes, 1,045 showing higher expression in males and 1,437 showing higher expression in females. In contrast to the widespread effects of the loss of STAT5b, STAT5a deficiency had a limited but well-defined impact on liver sex specificity, with 219 of 1,437 female-predominant genes (15%) specifically decreased in expression in STAT5a-deficient female liver. Analysis of liver RNAs from wild-type mice representing three mixed or outbred strains identified 1,028 sexually dimorphic genes across the strains, including 393 female-predominant genes, of which 89 (23%) required STAT5a for normal expression in female liver. These findings highlight the importance of STAT5a for regulation of sex-specific gene expression specifically in female liver, in striking contrast to STAT5b, whose major effects are restricted to male liver.
Ageing affects drugs metabolism influencing the therapeutic efficacy and safety of drugs. By using the experimental model of aged male rats, we investigated the influence of ageing on some CYP2C ...isoforms, the most important CYP450 sub-family in rats. The activity of the male specific CYP2C11 is decreased by 55% in senescent male rats. This correlates with a significant reduction of both protein content (80%) and mRNA (60%) indicating a demasculinization process. The expression of CYP2C12, a female specific isoform, is induced in senescent male rats indicating a feminization process. Neither the activity nor the expression of CYP2C6, a female predominant isoform, is modified in senescent male rats. Thereafter, certain putative GH mediators like some liver enriched transcription factors (LETFs) or STAT5b were investigated. The amount of HNF3beta mRNA, a transcription factor involved in the up-regulation of CYP2C12, has been shown to increase by about three-fold in senescent male rats. With regard to STAT5b, which has been reported to be involved in the male specific regulation of CYP2C11, large amounts of phosphorylated STAT5 were observed in the liver of senescent male rats. These results indicate that while the induction of CYP2C12 during ageing could be due, at least partially, to the enhanced HNF3beta expression, the decline of CYP2C11 is unlikely related to a decrease of STAT5 activation.
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