Agneta Mode

Hepatic steroid hydroxylating enzymes are controlled by the sexually dimorphic pattern of growth hormone secretion in normal and dwarf rats.

In rats, the onset of the sexually dimorphic pattern of growth hormone (GH) secretion and increased hepatic GH‐binding capacity at puberty are temporally correlated with the sex‐dependent expression of some hepatic cytochrome P450 enzymes involved in steroid metabolism. There are indications that the expression of the GH receptor gene itself is dependent on the sexually differentiated pattern of GH secretion. However, the molecular mechanisms by which a given pattern of GH secretion turns on a specific set of genes in the hepatocyte are not yet understood. Studies of the cytochrome P450 2C gene subfamily in hypophysectomized rats and isolated hepatocytes suggest that one major mechanism of GH action in the liver occurs through modulation of gene transcriptional initiation. The occurrence, in dwarf rats and in rats treated neonatally with monosodium glutamate, of sex differences in GH secretion and liver steroid metabolism typical of normal rats, in spite of a 95% reduction in pituitary GH levels, is compatible with the notion that extremely low levels of circulating GH are sufficient to regulate the expression of liver steroid‐metabolizing enzymes. This, together with the fact that single daily subcutaneous injections of GH are sufficient to masculinize the liver of a hypophysectomized rat, indicates that neither the amplitude nor the frequency of the GH pulse is recognized as male or female by the hepatocyte, but rather the complete and prolonged suppression (in males) or the persistence (in females) of circulating GH during the trough period after a GH surge.—Legraverend, C.; Mode, A.; Wells, T.; Robinson, I.; Gustafsson, J.‐A. Hepatic steroid hydroxylating enzymes are controlled by the sexually dimorphic pattern of growth hormone secretion in normal and dwarf rats. FASEB J. 6: 711‐718; 1992.

Ghrelin induces abdominal obesity via GHS-R-dependent lipid retention

Circulating ghrelin elevates abdominal adiposity by a mechanism independent of its central orexigenic activity. In this study we tested the hypothesis that peripheral ghrelin induces a depot-specific increase in white adipose tissue (WAT) mass in vivo by GH secretagogue receptor (GHS-R(1a))-mediated lipolysis. Chronic iv infusion of acylated ghrelin increased retroperitoneal and inguinal WAT volume in rats without elevating superficial sc fat, food intake, or circulating lipids and glucose. Increased retroperitoneal WAT mass resulted from adipocyte enlargement probably due to reduced lipid export (ATP-binding cassette transporter G1 mRNA expression and circulating free fatty acids were halved by ghrelin infusion). In contrast, ghrelin treatment did not up-regulate biomarkers of adipogenesis (peroxisome proliferator-activated receptor-gamma2 or CCAAT/enhancer binding protein-alpha) or substrate uptake (glucose transporter 4, lipoprotein lipase, or CD36) and although ghrelin elevated sterol-regulatory element-binding protein 1c expression, WAT-specific mediators of lipogenesis (liver X receptor-alpha and fatty acid synthase) were unchanged. Adiposity was unaffected by infusion of unacylated ghrelin, and the effects of acylated ghrelin were abolished by transcriptional blockade of GHS-R(1a), but GHS-R(1a) mRNA expression was similar in responsive and unresponsive WAT. Microarray analysis suggested that depot-specific sensitivity to ghrelin may arise from differential fine tuning of signal transduction and/or lipid-handling mechanisms. Acylated ghrelin also induced hepatic steatosis, increasing lipid droplet number and triacylglycerol content by a GHS-R(1a)-dependent mechanism. Our data imply that, during periods of energy insufficiency, exposure to acylated ghrelin may limit energy utilization in specific WAT depots by GHS-R(1a)-dependent lipid retention.

Regulation of sexual differentiation in drug and steroid metabolism

Certain members of the cytochrome P-450 family are expressed at different levels in the livers of male and female rats. Although little is known of the functional significance of these sex differences, progress has been made towards the understanding of the endocrine control of hepatic sex differences in cytochrome P-450 levels. Jan-Ake Gustafsson and colleagues describe a subpopulation of hepatic sexually differentiated P-450s that is regulated by sex differences in growth hormone (GH) secretory pattern. This secretory pattern is in turn regulated by gonadal steroids. These studies demonstrate a novel action of GH and suggest that the hormonal secretory rhythm is pivotal in determination of biological effects.

Sex and the Liver – A Journey Through Five Decades

Metabolism of steroids and drugs in rodents is sexually differentiated. The reason for this turned out to be the sexually differentiated growth hormone (GH) secretory pattern regulating the expression of a number of hepatic cytochrome P-450 genes. Although not fully resolved, it is clear that several signaling pathways and transcription factors are involved in mediating the effects of GH. It may be argued that such a well-controlled physiological system should have an important biological role and we speculate that the demands of a robust hepatic steroid metabolism during pregnancy has led to the development of this sexually differentiated hypothalamo-pituitary-liver axis.

Constitutive expression and hormonal regulation of male sexually differentiated cytochromes P450 in primary cultured rat hepatocytes

Experiments, predominantly performed in vivo, have shown that the pattern of growth hormone (GH) release from the pituitary gland is a major regulator of sex-specific cytochromes P450 (P450) in rats and other rodents. However, difficulty in constitutively expressing male-specific forms of P450 using in vitro models, such as primary cell culture, has impeded efforts to examine the direct actions of hormones on these enzyme forms. In the present study mRNA species for the male-specific P450 2C11 and 2C13, but not 3A2, were successfully expressed in primary hepatocytes cultured on a laminin-rich extracellular matrix (matrigel) in a serum-free, chemically defined medium containing insulin as the only hormone. When cells were exposed to GH (100 ng/ml), 2C11 mRNA expression was virtually abolished and 2C13 expression decreased to approximately 50% of control values, demonstrating that the negative regulation of these P450 forms by GH is a direct action on hepatocytes. Dexamethasone (DEX, 10(-8) M) increased the expression of 2C11 to 195% while decreasing 2C13 expression to 25% of control values. When GH and DEX were administered concurrently 2C11 was downregulated, indicating that GH is the dominant regulatory hormone for this form. L-Triiodothyronine (T3) (10(-9) M) suppressed 2C11 (46% of control) but had no effect on 2C13 mRNA expression. The positive regulatory effect of glucocorticoids on 2C11 was also found to occur in vivo and demonstrated to operate predominantly at the transcriptional level. This study demonstrates that primary cultures of hypatocytes are a suitable in vitro model for studies on regulation of some male-specific P450 forms and that GH, DEX, and T3 act directly on hepatocytes at a pretranslational level to regulate these forms.

Desensitization of the Growth Hormone-Induced Janus Kinase 2 (Jak 2)/Signal Transducer and Activator of Transcription 5 (Stat5)-Signaling Pathway Requires Protein Synthesis and Phospholipase C1

Signal transducers and activators of transcription (Stat) proteins are latent cytoplasmic transcription factors that are tyrosine phosphorylated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line stably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Stat5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a desensitization characterized by 1) a rapid decrease in Stat5 DNA-binding activity. The rate of decrease of activity was rapid up to 1 h of GH treatment, and the remaining activity declined slowly thereafter. The activity of Stat5 present after 5 h is still higher than the control levels and almost 10–20% with respect to ...

Physiological Differences between Human and Rat Primary Hepatocytes in Response to Liver X Receptor Activation by 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic Acid Hydrochloride (GW3965)

The liver is central to the maintenance of glucose and lipid homeostasis, and liver X receptors (LXRs) are key regulators of expression of the genes involved. So far, effects of activation of LXR in human hepatocytes have not been well characterized. Here we show that treatment of primary human hepatocytes with the synthetic LXR ligand 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic acid hydrochloride (GW3965) results in reduced output of bile acids and very low density lipoprotein triglycerides and induced expression of adipose differentiation-related protein accompanied by increased lipid storage. Genome wide-expression profiling identified novel human LXR target genes in the glycolytic and lipogenic pathways and indicated that LXR activation reduced hepatic insulin sensitivity. Comparative experiments showed significant differences in the response to GW3965 between human and rat hepatocytes, raising the question as to how well rodent models reflect the human situation. In summary, the risk of hepatic steatosis upon pharmaceutical targeting of LXR may be a particularly serious consequence in humans.

Growth hormone regulation of hepatic cytochrome P450 expression in the rat

GH by means of its sexually differentiated secretory pattern is the predominant regulator of the expression of cytochrome P450 enzymes responsible for a sexual dimorphism of hepatic steroid metabolism. Other hormones, such as gonadal, thyroid and glucocorticoid hormones, as well as insulin appear to modulate the sexually differentiated expression of these enzymes. The major constitutively expressed sex specific forms of P450, belonging to the P4502C-subfamily, have been shown to be regulated by GH at the level of transcription. However, the GH postreceptor events leading to increased or decreased transcriptional activity are essentially unknown. Neither is the functional role of the soluble GH binding protein yet resolved. On-going protein synthesis is a prerequisite for GH transcriptional activation of the female specific P4502C12 but not for all GH effects in the hepatocyte. With regard to signalling mechanisms PKC activity appears to be permissive for the GH induction of P4502C12 but some as yet unidentified factor/kinase(s) may also be activated. The transcriptional control exerted on the rat P4502C-gene subfamily by the pattern of GH secretion offers a versatile tool to elucidate the molecular mechanisms of GH regulation of cytochrome P450 expression.

Transcriptional activity and developmental expression of liver X receptor (lxr) in Zebrafish

Mammalian liver‐X‐receptors (LXRs) are transcription factors activated by oxysterols. They play an essential role in lipid and glucose metabolism. We have cloned the open reading frame of zebrafish lxr and describe its genomic organization. Zebrafish lxr encodes a 50‐kDa protein with high sequence similarity to mammalian LXRα. In transfection assays, the encoded protein showed transcriptional activity in response to LXR‐ligands. Treatment of adult zebrafish with the synthetic LXR ligand, GW3965, induced expression of genes involved in hepatic cholesterol and lipid pathways. Using qPCR and in situ hybridization, we found ubiquitous expression of lxr mRNA during the first 24 hr of development, followed by more restricted expression, particularly to the liver at 3dpf and the liver and intestine at 4dpf. In adult fish, all examined organs expressed lxr. In addition to a metabolic role of lxr, the temporal expression pattern suggests a developmental role in, e.g., the liver and CNS. Developmental Dynamics 237:1090–1098, 2008.

Cis desensitizes GH induced Stat5 signaling in rat liver cells

Recently a novel family of proteins, the Cis/Socs family, has been shown to constitute negative regulators of cytokine-induced Jak/Stat signaling. Here we demonstrate that Socs-2 and Cis mRNA expression in rat liver is dependent on the presence of growth hormone (GH), and that GH induce Cis mRNA expression in cultures of primary rat hepatocytes. Furthermore, cotransfection studies in the rat liver cell line, BRL-4, revealed that constitutive expression of Cis, but not Socs-2, inhibited the GH-induced transactivation of a Stat5-responsive reporter gene construct. This indicates a functional role for Cis in the desensitization of GH activated Jak/Stat5 signaling in rat liver cells. In response to the intermittent pattern of GH secretion in male rats, GH activates Stat5b signaling whereas this activation is blunted in female rats having a continuous pattern of GH secretion. We hypothesize that GH induction of Cis could be one mechanism by which sexually dimorphic GH signaling via Stat5b is achieved in the rat liver.