Tissue-specific knockouts of steroidogenic factor 1

Targeted gene disruption has produced knockout (KO) mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 KO mice lacked adrenal glands and gonads, and also had impaired expression of gonadotropins in pituitary gonadotropes and marked structural abnormalities of the ventromedial hypothalamic nucleus (VMH). To define the roles of SF-1 within discrete sites of the hypothalamic-pituitary-steroidogenic organ axis, we have sought to make tissue-specific SF-1 KO mice (as reviewed here). We first used adrenal transplants to restore adrenal function in global SF-1 KO mice, providing a physiological form of a "VMH-specific" KO to study the roles of SF-1 in weight regulation. These adrenal-transplanted SF-1 KO mice became obese due to decreased locomotor activity, providing a novel model of hypothalamic obesity. Mice with a pituitary-specific KO of SF-1 mediated by the Cre-loxP recombination strategy exhibited hypogonadotropic hypogonadism, revealing essential roles of SF-1 in pituitary function in vivo. Ongoing studies seek to inactivate SF-1 in the brain or specific gonadal cell types, thereby defining its roles in development and function at these sites. In addition, we review our use of bacterial artificial chromosome transgenesis to develop a fluorescent marker for cells that express SF-1.     

Selective loss of leptin receptors in the ventromedial hypothalamic nucleus results in increased adiposity and a metabolic syndrome

Leptin, an adipocyte-derived hormone, has emerged as a critical regulator of energy homeostasis. The leptin receptor (Lepr) is expressed in discrete regions of the brain; among the sites of highest expression are several mediobasal hypothalamic nuclei known to play a role in energy homeostasis, including the arcuate nucleus, the ventromedial hypothalamic nucleus (VMH), and the dorsomedial hypothalamic nucleus. Although most studies have focused on leptin's actions in the arcuate nucleus, the role of Lepr in these other sites has received less attention. To explore the role of leptin signaling in the VMH, we used bacterial artificial chromosome transgenesis to target Cre recombinase to VMH neurons expressing steroidogenic factor 1, thereby inactivating a conditional Lepr allele specifically in steroidogenic factor 1 neurons of the VMH. These knockout (KO) mice, designated Lepr KO(VMH), exhibited obesity, particularly when challenged with a high-fat diet. On a low-fat diet, Lepr KO(VMH) mice exhibited significantly increased adipose mass even when their weights were comparable to wild-type littermates. Furthermore, these mice exhibited a metabolic syndrome including hepatic steatosis, dyslipidemia, and hyperleptinemia. Lepr KO(VMH) mice were hyperinsulinemic from the age of weaning and eventually developed overt glucose intolerance. These data define nonredundant roles of the Lepr in VMH neurons in energy homeostasis and provide a model system for studying other actions of leptin in the VMH.     

Steroidogenic factor 1 (SF-1) is essential for ovarian development and function

The orphan nuclear receptor steroidogenic factor 1 (SF-1) was identified originally as a key regulator of the tissue-specific expression of the cytochrome P450 steroid hydroxylases. Hints at considerably broader roles for SF-1 came from analyses of its expression pattern in mouse embryos. As anticipated, SF-1 was expressed in the adrenal glands and gonads from their early stages of development. Surprisingly, SF-1 also was expressed outside of the primary steroidogenic tissues in the anterior pituitary and hypothalamus. SF-1 knockout mice dramatically confirmed its multiple essential roles in vivo. These mice lacked adrenal glands and gonads, leading to adrenocortical insufficiency and male-to-female sex reversal of their internal and external genitalia. SF-1 knockout mice also had impaired pituitary expression of gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-gonadal axis. With some focus on the ovary, this review summarizes experiments that have defined essential roles of SF-1 in endocrine development, and highlights important areas for future studies.     

SF-1: a critical mediator of steroidogenesis

Studies in knockout mice have established that the orphan nuclear receptor steroidogenic factor 1 (SF-1) plays essential roles in the development and function of the primary steroidogenic organs. These SF-1 knockout mice lacked adrenal glands and gonads, causing adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of pituitary gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-steroidogenic organ axis. Ongoing experiments are directed at developing methods to inactivate SF-1 in a tissue-specific manner.     

Approaches to define the role of SF-1 at different levels of the hypothalamic-pituitary-steroidogenic organ axis.

Targeted gene disruption has produced knockout mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 knockout mice lacked adrenal glands and gonads, resulting in adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of pituitary gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at multiple levels of the hypothalamic-pituitary-steroidogenic tissue axis. Using the Cre-loxP system, we now have generated mice in which SF-1 is inactivated selectively in the anterior pituitary. These pituitary-specific SF-1 knockout mice were sterile and failed to exhibit sexual maturation. Histologically, their gonads were markedly hypoplastic, weighing only approximately 5% of the gonads of wild-type mice. Consistent with an important role of SF-1 in gonadotropes, there were no cells in the pituitary gland that expressed either follicle-stimulating hormone (FSH) or luteinizing hormone (LH). These pituitary-specific SF-1 knockout mice are a novel genetic model of hypogonadotropic hypogonadism and establish essential roles of SF-1 in gonadotropin expression.     

Pituitary-specific knockout of steroidogenic factor 1.

Knockout mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1) revealed its essential roles at multiple levels of endocrine development and function. These SF-1 knockout mice lacked adrenal glands and gonads, thereby manifesting adrenal insufficiency and sex reversal of their internal and external genitalia. Their pituitary gonadotropes failed to express several markers of normal differentiated function, and they lacked a specific hypothalamic nucleus, the ventromedial hypothalamic nucleus (VMH). Using the Cre-loxP system, we generated mice whose gene encoding SF-1 was inactivated specifically in the anterior pituitary. These pituitary-specific SF-1 knockout mice were sterile and never matured sexually. Their gonads weighed only approximately 5% of the weight of wild-type gonads. SF-1 immunoreactivity was absent in the anterior pituitary but was unaffected in the adrenal cortex, validating the selectivity of the gene targeting strategy. Consistent with an important role of SF-1 in gonadotropes, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were markedly decreased in the pituitary-specific SF-1 knockout mice. The pituitary-specific SF-1 knockout mice are a novel genetic model of hypogonadotropic hypogonadism and establish essential roles of SF-1 in gonadotropin expression.     

Approaches to define the role of SF-1 at different levels of the hypothalamic-pituitary-steroidogenic organ axis

Targeted gene disruption has produced knockout mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 knockout mice lacked adrenal glands and gonads and consequently exhibited adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of gonadtropins in the anterior pituitary gonadotropes and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-steroidogenic organ axis. Ongoing experiments are directed at using evolving techniques for tissue-specific gene inactivation to define the roles of SF-1 within discrete sites of the hypthalamic-pituitary-steroidogenic organ axis.     

Regulatory functions of limbic Y1 receptors in body weight and anxiety uncovered by conditional knockout and maternal care

Neuropeptide Y (NPY) plays an important role in stress, anxiety, obesity, and energy homeostasis via activation of NPY-Y1 receptors (Y1Rs) in the brain. However, global knockout of the Npy1r gene has low or no impact on anxiety and body weight. To uncover the role of limbic Y1Rs, we generated conditional knockout mice in which the inactivation of the Npy1r gene was restricted to excitatory neurons of the forebrain, starting from juvenile stages (Npy1r(rfb)). Npy1r(rfb) mice exhibited increased anxiety and reduced body weight, less adipose tissue, and lower serum leptin levels. Npy1r(rfb) mutants also had a hyperactive hypothalamic-pituitary-adrenocortical axis, as indicated by higher peripheral corticosterone and higher density of NPY immunoreactive fibers and corticotropin releasing hormone immunoreactive cell bodies in the paraventricular hypothalamic nucleus. Importantly, through fostering experiments, we determined that differences in phenotype between Npy1r(rfb) and Npy1r(2lox) mice became apparent when both genotypes were raised by FVB/J but not by C57BL/6J dams, suggesting that limbic Y1Rs are key targets of maternal care-induced programming of anxiety and energy homeostasis.     

The roles of the nuclear receptor steroidogenic factor 1 in endocrine differentiation and development.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) has emerged as a critical determinant of adrenal and gonadal differentiation, development, and function. SF-1 was initially isolated as a positive regulator of the cytochrome P450 steroid hydroxylases in the adrenal glands and gonads; developmental analyses subsequently showed that SF-1 was also expressed in the diencephalon and anterior pituitary, suggesting additional roles in endocrine function. Analyses of knockout mice deficient in SF-1 revealed multiple abnormalities, including adrenal and gonadal agenesis, male to female sex reversal of the internal genitalia, impaired gonadotrope function, and absence of the ventromedial hypothalamic nucleus. Taken together, these results implicate SF-1 as a global regulator within the hypothalamic-pituitary-gonadal axis and the adrenal cortex.     

Knockout mice lacking steroidogenic factor 1 are a novel genetic model of hypothalamic obesity.

Knockout (KO) mice lacking steroidogenic factor 1 (SF-1) exhibit a phenotype that includes adrenal and gonadal agenesis, impaired gonadotropin expression, and abnormalities of the ventromedial hypothalamic nucleus (VMH). Studies in rodents with lesions of the ventromedial hypothalamus have implicated the VMH in body weight regulation, suggesting that SF-1 KO mice may provide a genetic model of obesity. To prevent death, SF-1 KO mice were rescued with corticosteroid injections, followed by syngeneic adrenal transplants from wild-type (WT) littermates. Corticosterone and ACTH levels in WT and SF-1 KO mice were indistinguishable, documenting restoration of hypothalamic-pituitary-adrenal function. Although weights at earlier ages did not differ significantly from WT littermates, SF-1 KO mice were significantly heavier by 8 wk of age and eventually weighed almost twice as much as WT controls. Obesity in SF-1 KO mice predominantly resulted from decreased activity rather than increased food intake. Leptin was increased markedly, insulin was modestly elevated, and glucose was indistinguishable from WT mice. Although sex steroids in rodents affect weight, ovariectomy did not abolish the weight difference between WT and SF-1 KO mice. These SF-1 KO mice are a genetic model of late-onset obesity that may help elucidate the role of the VMH in weight regulation.     

Development of a transgenic green fluorescent protein lineage marker for steroidogenic factor 1

Knockout (KO) mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired function of pituitary gonadotropes, and abnormalities of the ventromedial hypothalamic nucleus (VMH). To develop a lineage marker for cells that express SF-1, we used bacterial artificial chromosome (BAC) transgenesis. A BAC fragment containing 50 kb of the mouse Nr5a1 gene was placed upstream of the coding sequence for enhanced green fluorescent protein (eGFP) and used to generate SF-1/eGFP transgenic mice. These sequences directed eGFP expression to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, VMH neurons, and reticuloendothelial cells of the spleen. Despite the essential role of SF-1 in gonadotropes, eGFP was not expressed in the anterior pituitary. These studies show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgene provides a valuable tool to expand our understanding of the actions of SF-1 in endocrine development and function.     

Role of the multidrug resistance protein-1 (MRP1) for endothelial progenitor cell function and survival

The multidrug resistance related protein-1 (MRP1) is a member of the ATP binding cassette (ABC) of cell surface transport proteins expressed in multiple cell lines and tissues including endothelial cells and haematopoietic stem cells. MRP1 blockade has been shown to prevent endothelial cell apoptosis and improve endothelial function. Besides mature endothelial cells vascular homing of endothelial progenitor cells (EPC) contributes to endothelial regeneration after vascular damage. Thus, we hypothesized that MRP1 influences number and function of EPCs and mechanisms of vascular repair. To test this, we investigated the effects of MRP1 inhibition in vitro and in vivo. MRP1 is abundantly expressed in cultured human early outgrowth EPCs. Pharmacological inhibition of MRP1 by MK571 increased intracellular glutathione levels and reduced intracellular reactive oxygen species levels. This stabilization of the intracellular redox homeostasis via inhibition of MRP1 prevented angiotensin II-induced apoptosis and increased the number of early outgrowth EPCs and colony forming units in vitro. To extend the observed cytoprotective effect of MRP1 blockade in EPCs to an in vivo situation, MRP1^−/− knockout mice were investigated. MRP1^−/− knockout mice showed significantly increased numbers of EPCs circulating in the peripheral blood and residing in the bone marrow. Consistently, colony forming unit formation was enhanced and rate of apoptosis reduced in early outgrowth EPCs derived from MRP1^−/− knockout mice. In addition, MRP1^−/− knockout mice showed improved reendothelialization after carotid artery injury, and transfusion of MNCs derived from MRP1^−/− knockout mice into wild-type mice accelerated reendothelialization compared to transfusion of wild-type cells. These findings indicate that the enhanced function and survival of EPCs in MRP1^−/− knockout mice resulted in improved reendothelialization. In conclusion, MRP1 negatively influences EPC function and survival via perturbation of the intracellular redox homeostasis which finally leads to increased cellular apoptosis. These results reveal novel mechanistic insights and may identify MRP1 as therapeutic target to improve reendothelialization after vascular damage.     

Endothelial nitric oxide synthase gene transfer restores endothelium–dependent relaxations and attenuates lesion formation in carotid arteries in apolipoprotein E–deficient mice

Nitric oxide (NO) and monocyte chemoattractant protein–1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP–1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP–1 expression. We have evaluated adenovirus–mediated gene transfer of human endothelial NO synthase (eNOS) and of a N–terminal deletion (8ND) mutant of the MCP–1 gene that acts as a MCP–1 inhibitor in arteriosclerosis–prone, apolipoprotein E-deficient (ApoE^–/–) mice. Endotheliumdependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5–week follow–up (p < 0.05). In contrast, 8ND–MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium–dependent vasodilation and inhibits lesion formation in ApoE^–/– mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long–term efficacy of eNOS gene therapy for primary arteriosclerosis.     

Evidence that oestrogen receptor-α plays an important role in the regulation of glucose homeostasis in mice: insulin sensitivity in the liver

Aims/hypothesis We used oestrogen receptor-α (ERα) knockout (ERKO) and receptor-β (ERβ) knockout (BERKO) mice to investigate the mechanism(s) behind the effects of oestrogens on glucose homeostasis. Methods Endogenous glucose production (EGP) was measured in ERKO mice using a euglycaemic–hyperinsulinaemic clamp. Insulin secretion was determined from isolated islets. In isolated muscles, glucose uptake was assayed by using radiolabelled isotopes. Genome-wide expression profiles were analysed by high-density oligonucleotide microarray assay, and the expression of the genes encoding steroyl-CoA desaturase and the Leptin receptor (Scd1 and Lepr, respectively) was confirmed by RT-PCR. Results ERKO mice had higher fasting blood glucose, plasma insulin levels and IGT. The plasma leptin level was increased, while the adiponectin concentration was decreased in ERKO mice. Levels of both glucose- and arginine-induced insulin secretion from isolated islets were similar in ERKO and wild-type mice. The euglycaemic–hyperinsulinaemic clamp revealed that suppression of EGP by increased insulin levels was blunted in ERKO mice, which suggests a pronounced hepatic insulin resistance. Microarray analysis revealed that in ERKO mice, the genes involved in hepatic lipid biosynthesis were upregulated, while genes involved in lipid transport were downregulated. Notably, hepatic Lepr expression was decreased in ERKO mice. In vitro studies showed a modest decrease in insulin-mediated glucose uptake in soleus and extensor digitorum longus (EDL) muscles of ERKO mice. BERKO mice demonstrated normal glucose tolerance and insulin release. Conclusions/interpretation We conclude that oestrogens, acting via ERα, regulate glucose homeostasis mainly by modulating hepatic insulin sensitivity, which can be due to the upregulation of lipogenic genes via the suppression of Lepr expression.