Bone morphogenic protein 9 is a novel thermogenic hepatokine secreted in response to cold exposure.

OBJECTIVE: Maintaining a constant core body temperature is essential to homeothermic vertebrate survival. Adaptive thermogenesis in brown adipose tissue and skeletal muscle is the primary mechanism of adjustment to an external stimulus such as cold exposure. Recently, several reports have revealed that the liver can play a role as a metabolic hub during adaptive thermogenesis. In this study, we suggest that the liver plays a novel role in secreting thermogenic factors in adaptive thermogenesis. Bone morphogenetic protein 9 (BMP9) is a hepatokine that regulates many biological processes, including osteogenesis, chondrogenesis, hematopoiesis, and angiogenesis. Previously, BMP9 was suggested to affect preadipocyte proliferation and differentiation. However, the conditions and mechanisms underlying hepatic expression and secretion and adipose tissue browning of BMP9 remain largely unknown. In this study, we investigated the physiological conditions for secretion and the regulatory mechanism of hepatic Bmp9 expression and the molecular mechanism by which BMP9 induces thermogenic gene program activation in adipose tissue. Here, we also present the pharmacological effects of BMP9 on a high-fat-induced obese mouse model. METHODS: To investigate the adaptive thermogenic role of BMP9 in vivo, we challenged mice with cold temperature exposure for 3 weeks and then examined the BMP9 plasma concentration and hepatic expression level. The cellular mechanism of hepatic Bmp9 expression under cold exposure was explored through promoter analysis. To identify the role of BMP9 in the differentiation of brown and beige adipocytes, we treated pluripotent stem cells and inguinal white adipose tissue (iWAT)-derived stromal-vascular (SV) cells with BMP9, and brown adipogenesis was monitored by examining thermogenic gene expression and signaling pathways. Furthermore, to evaluate the effect of BMP9 on diet-induced obesity, changes in body composition and glucose tolerance were analyzed in mice administered recombinant BMP9 (rBMP9) for 8 weeks. RESULTS: Hepatic Bmp9 expression and plasma levels in mice were significantly increased after 3 weeks of cold exposure. Bmp9 mRNA expression in the liver was regulated by transcriptional activation induced by cAMP response-element binding protein (CREB) and CREB-binding protein (CBP) on the Bmp9 promoter. Treatment with BMP9 promoted the differentiation of multipotent stem cells and iWAT-derived SV cells into beige adipocytes, as indicated by the increased expression of brown adipocyte and mitochondrial biogenesis markers. Notably, activation of the mothers against decapentaplegic homolog 1 (Smad1) and p44/p42 mitogen-activated protein kinase (MAPK) pathways was required for the induction of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression in BMP9-induced differentiation of SVs into beige adipocytes. The administration of rBMP9 in vivo also induced browning markers in white adipose tissue. In high-fat diet-induced obese mice, rBMP9 administration conferred protection against obesity and enhanced glucose tolerance. CONCLUSIONS: BMP9 is a hepatokine regulated by cold-activated CREB and CBP and enhances glucose and fat metabolism by promoting the activation of the thermogenic gene program in adipocytes. These data implicate BMP9 as a potential pharmacological tool for protecting against obesity and type 2 diabetes.

Glutamate dehydrogenase as a neuroprotective target against brain ischemia and reperfusion.

Deregulation of glutamate homeostasis is associated with degenerative neurological disorders. Glutamate dehydrogenase (GDH) is important for glutamate metabolism and plays a central role in expanding the pool of tricarboxylic acid (TCA) cycle intermediate alpha-ketoglutarate (α-KG), which improves overall bioenergetics. Under high energy demand, maintenance of ATP production results in functionally active mitochondria. Here, we tested whether the modulation of GDH activity can rescue ischemia/reperfusion-induced neuronal death in an in vivo mouse model of middle artery occlusion and an in vitro oxygen/glucose depletion model. Iodoacetate, an inhibitor of glycolysis, was also used in a model of energy failure, remarkably depleting ATP and α-KG. To stimulate GDH activity, the GDH activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid and potential activator beta-lapachone were used. The GDH activators restored α-KG and ATP levels in the injury models and provided potent neuroprotection. We also found that beta-lapachone increased glutamate utilization, accompanied by a reduction in extracellular glutamate. Thus, our hypothesis that mitochondrial GDH activators increase α-KG production as an alternative energy source for use in the TCA cycle under energy-depleted conditions was confirmed. Our results suggest that increasing GDH-mediated glutamate oxidation represents a new therapeutic intervention for neurodegenerative disorders, including stoke.

A potent and selective 11ß-hydroxysteroid dehydrogenase type 1 inhibitor, SKI2852, ameliorates metabolic syndrome in diabetic mice models.

11ß-Hydroxysteroid dehydrogenase type 1 (11ßHSD1) has been targeted for new drugs to treat type 2 diabetes and metabolic syndrome. In this study, we determined whether the inhibition of 11ßHSD1 with a new selective inhibitor, SKI2852, could improve lipid profiles, glucose levels, and insulin sensitivity in type 2 diabetic and obese conditions. SKI2852 showed a potent inhibition of cortisone to cortisol conversion for over 80% in both liver and adipose tissue ex vivo from orally administered C57BL/6 mice, and in vivo analysis results were consistent with this. Repeated oral administrations of SKI2852 in diet-induced obesity (DIO) and ob/ob mice revealed a partially beneficial effect of SKI2852 in improving levels of cholesterols, triglycerides, free fatty acids, postprandial glucose, and/or blood hemoglobinA1c. SKI2852 significantly reduced body weight increase in ob/ob mice, and efficiently suppressed hepatic mRNA levels of gluconeogenic enzymes in DIO mice. Moreover, SKI2852 enhanced hepatic and whole body insulin sensitivities in hyperinsulinemic-euglycemic clamp experiment in DIO mice. In conclusion, these results indicate that selective and potent inhibition of 11ßHSD1 by SKI2852, thus blockade of active glucocorticoid conversion, may improve many aspects of metabolic parameters in type 2 diabetes and metabolic diseases, mainly by inhibitions of hepatic gluconeogenesis and partial improvements of lipid profiles. Our study strongly support that SKI2852 may have a great potential as a novel candidate drug for the treatment of diabetes and metabolic diseases.

Insulin resistance and white adipose tissue inflammation are uncoupled in energetically challenged Fsp27-deficient mice.

Fsp27 is a lipid droplet-associated protein almost exclusively expressed in adipocytes where it facilitates unilocular lipid droplet formation. In mice, Fsp27 deficiency is associated with increased basal lipolysis, 'browning' of white fat and a healthy metabolic profile, whereas a patient with congenital CIDEC deficiency manifested an adverse lipodystrophic phenotype. Here we reconcile these data by showing that exposing Fsp27-null mice to a substantial energetic stress by crossing them with ob/ob mice or BATless mice, or feeding them a high-fat diet, results in hepatic steatosis and insulin resistance. We also observe a striking reduction in adipose inflammation and increase in adiponectin levels in all three models. This appears to reflect reduced activation of the inflammasome and less adipocyte death. These findings highlight the importance of Fsp27 in facilitating optimal energy storage in adipocytes and represent a rare example where adipose inflammation and hepatic insulin resistance are disassociated.

In-depth metabolic phenotyping of genetically engineered mouse models in obesity and diabetes.

The world-wide prevalence of obesity and diabetes has increased sharply during the last two decades. Accordingly, the metabolic phenotyping of genetically engineered mouse models is critical for evaluating the functional roles of target genes in obesity and diabetes, and for developing new therapeutic targets. In this review, we discuss the practical meaning of metabolic phenotyping, the strategy of choosing appropriate tests, and considerations when designing and performing metabolic phenotyping in mice.

ß-Lapachone alleviates alcoholic fatty liver disease in rats.

UNLABELLED: Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is altered. It is thought that altered NAD(+)/NADH redox potential by alcohol in the liver causes fatty liver by inhibiting fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. ß-Lapachone (ßL), a naturally occurring quinone, has been shown to stimulate fatty acid oxidation in an obese mouse model by activating adenosine monophosphate-activated protein kinase (AMPK). In this report, we clearly show that ßL reduced alcohol-induced hepatic steatosis and induced fatty acid oxidizing capacity in ethanol-fed rats. ßL treatment markedly decreased hepatic lipids while serum levels of lipids and lipoproteins were increased in rats fed ethanol-containing liquid diets with ßL administration. Furthermore, inhibition of lipolysis, enhancement of lipid mobilization to mitochondria and upregulation of mitochondrial ß-oxidation activity in the soleus muscle were observed in ethanol/ßL-treated animals compared to the ethanol-fed rats. In addition, the activity of alcohol dehydrogenase, but not aldehyde dehydrogenase, was significantly increased in rats fed ßL diets. ßL-mediated modulation of NAD(+)/NADH ratio led to the activation of AMPK signaling in these animals. CONCLUSION: Our results suggest that improvement of fatty liver by ßL administration is mediated by the upregulation of apoB100 synthesis and lipid mobilization from the liver as well as the direct involvement of ßL on NAD(+)/NADH ratio changes, resulting in the activation of AMPK signaling and PPARα-mediated ß-oxidation. Therefore, ßL-mediated alteration of NAD(+)/NADH redox potential may be of potential therapeutic benefit in the clinical setting.

Assessment of ZnO and SiO2 nanoparticle permeability through and toxicity to the blood-brain barrier using Evans blue and TEM.

As increasing variants of nanoparticles (NPs) are being used in various products, it has become apparent that size alone can no longer adequately explain the variety of generated toxic profiles. Recent studies with NPs have suggested that various sizes of NPs could determine in vitro toxicity. In an attempt to address concerns regarding neurotoxicity of zinc oxide (ZnO) and silica (SiO2) NPs, these were examined after exposing them via oral, dermal, and intravenous administrations of NPs and their toxicological effects on the brain over a prescribed period of time were assessed. After 28 days of repeated oral administrations of ZnO or SiO2 independently, possibly due to damages to the blood brain barrier (BBB), neurotoxicity, were investigated by Evans blue technique. Next, in order to assess whether ZnO NPs could compromise the BBB, ZnO NPs were intravenously injected on day 0, 7, 14, 21 and 28 no further treatment was administered for 62 days. Deposition of SiO2 in brain from repeated dermal and oral administrations for 90 days were evaluated by transmission electron microscopy coupled with scanning energy-dispersive X-ray spectroscopy. Physiochemical profiles were principally determined on particle size at the beginning of the current toxicity investigations on ZnO and SiO2 NPs. The BBB was found to be intact after independent repeated oral administrations of ZnO or SiO2 NPs for 28 days, suggesting no significant damage. Neuronal death was also not observed after the intravenous administrations of ZnO NPs. After 90 days of repeated dermal and oral administration of SiO2 NPs, no deposition of NPs was observed in hippocampus, striatum, and cerebellum regions using transmission electron microscope analyses. These observations suggest that the BBB was not compromised and was able to block penetration of ZnO and SiO2 NPs, resulting in significant neurotoxic effects. Moreover, absence of SiO2 in three regions of brain after dermal and oral administrations for 90 days suggested that brain was protected from SiO2. No behavior change was observed in all studies, suggesting that 90 days may not be long enough to assess full neurotoxicity of NPs in vivo.

Enhanced activation of NAD(P)H: quinone oxidoreductase 1 attenuates spontaneous hypertension by improvement of endothelial nitric oxide synthase coupling via tumor suppressor kinase liver kinase B1/adenosine 5'-monophosphate-activated protein kinase-mediated guanosine 5'-triphosphate cyclohydrolase 1 preservation.

AIMS: Guanosine 5'-triphosphate cyclohydrolase-1 (GTPCH-1) is a rate-limiting enzyme in de-novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) coupling. Adenosine 5'-monophosphate-activated protein kinase (AMPK) is crucial for GTPCH-1 preservation, and tumor suppressor kinase liver kinase B1 (LKB1), an upstream kinase of AMPK, is activated by NAD-dependent class III histone deacetylase sirtuin 1 (SIRT1)-mediated deacetylation. ß-Lapachone has been shown to increase cellular NAD/NADH ratio via NAD(P)H: quinone oxidoreductase 1 (NQO1) activation. In this study, we have evaluated whether ß-lapachone-induced NQO1 activation modulates blood pressure (BP) through preservation of GTPCH-1 in a hypertensive animal model. METHODS AND RESULTS: Spontaneously hypertensive rats (SHRs), primary aortic endothelial cells, and endothelial cell line were used to investigate the hypotensive effect of ß-lapachone and its action mechanism. ß-Lapachone treatment dramatically lowered BP and vascular tension in SHRs and induced eNOS activation in endothelial cells. Consistent with these effects, ß-lapachone treatment also elevated levels of both aortic cGMP and plasma nitric oxide in SHRs. Meanwhile, ß-lapachone-treated SHRs showed significantly increased levels of aortic NAD, LKB1 deacetylation, and AMPK Thr phosphorylation followed by increased GTPCH-1 and tetrahydrobiopterin/dihydrobiopterin ratio. In-vitro study revealed that AMPK inhibition by overexpression of dominant-negative AMPK nearly abolished GTPCH-1 protein conservation. Enhanced LKB1 deacetylation and AMPK activation were also elicited by ß-lapachone in endothelial cells. However, inhibition of LKB1 deacetylation by blocking of NQO1 or SIRT1 blunted AMPK activation by ß-lapachone. CONCLUSION: This is the first study demonstrating that eNOS coupling can be regulated by NQO1 activation via LKB1/AMPK/GTPCH-1 modulation, which is possibly correlated with relieving hypertension. These findings provide strong evidence to suggest that NQO1 might be a new therapeutic target for hypertension.

NQO1 activation regulates angiotensin-converting enzyme shedding in spontaneously hypertensive rats.

AIMS: Angiotensin-converting enzyme (ACE) plays a key role in blood pressure (BP) homeostasis via regulation of angiotensin II. Active ACE ectodomain is enzymatically cleaved and released into body fluids, including plasma, and elevated plasma ACE levels are associated with increased BP. ß-lapachone (ßL) has been shown to increase cellular NAD(+)/NADH ratio via activation of NAD(P)H:quinone oxidoreductase 1 (NQO1). In this study, we evaluated whether NQO1 activation by ßL modulates BP through regulation of ACE shedding in an animal model of hypertension. METHODS AND RESULTS: Spontaneously hypertensive rats (SHR) and a human ACE-overexpressing rat lung microvascular endothelial cell line (RLMVEC-hACE) were used to investigate the mechanism by which ßL exerts a hypotensive effect. In vitro studies revealed that ßL significantly increased intracellular Ca(2+) ([Ca(2+)]i) levels and CaMKII Thr(286) phosphorylation, followed by diminished ACE cleavage secretion into culture media. Inhibition of ßL-induced [Ca(2+)]i level changes through intracellular Ca(2+) chelation, Nqo1-specific siRNA or ryanodine receptor blockade abolished not only ßL-induced increase in [Ca(2+)]i levels and CaMKII phosphorylation, but also ßL-mediated decrease in ACE shedding. The effect of ßL on ACE shedding was also blocked by inhibition of CaMKII. In SHR, ßL reduced BP following increase of CaMKII Thr(286) phosphorylation in the lung and decrease of ACE activity and angiotensin II levels in plasma. CONCLUSION: This is the first study demonstrating that ACE shedding is regulated by NQO1 activation, which is possibly correlated with relieving hypertension in SHR. These findings provide strong evidence suggesting that NQO1 might be a new target for ACE modulation and BP control.

AMPK activation with glabridin ameliorates adiposity and lipid dysregulation in obesity.

In this study, we demonstrate that activation of AMP-activated protein kinase (AMPK) with glabridin alleviates adiposity and hyperlipidemia in obesity. In several obese rodent models, glabridin decreased body weight and adiposity with a concomitant reduction in fat cell size. Further, glabridin ameliorated fatty liver and plasma levels of triglyceride and cholesterol. In accordance with these findings, glabridin suppressed the expression of lipogenic genes such as sterol regulatory element binding transcription factor (SREBP)-1c, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD)-1 in white adipose tissues and liver, whereas it elevated the expression of fatty acid oxidation genes such as carnitine palmitoyl transferase (CPT)1, acyl-CoA oxidase (ACO), and peroxisome proliferator-activated receptor (PPAR)α in muscle. Moreover, glabridin enhanced phosphorylation of AMPK in muscle and liver and promoted fatty acid oxidation by modulating mitochondrial activity. Together, these data suggest that glabridin is a novel AMPK activator that would exert therapeutic effects in obesity-related metabolic disorders.

Corpus cavernosal smooth muscle relaxation effect of a novel AMPK activator, beta-lapachone.

INTRODUCTION: Adenosine monophosphate-activated protein kinase (AMPK) activation is suggested to relax smooth muscle by endothelial nitric oxide synthase (eNOS) phosphorylation. AIM: To assess the mechanism and effect of a novel AMPK activator, beta-lapachone, upon cavernosal smooth muscle relaxation and the therapeutic potential for erectile dysfunction. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with beta-lapachone. The lysates were blotted with specific antibodies for phosphorylated AMPK (p-AMPK) or phosphorylated eNOS (p-eNOS). The membranes were re-blotted for total AMP total eNOS, or beta-actin. The eNOS activity was measured by the conversion of L-14C-arginine to L-14C-citrulline in HUVECs lysates. In a separated experiment, cavernosal strips from New Zealand white rabbits were harvested for organ bath study and the relaxation effect of beta-lapachone on phenylephrine-induced contracted strips was evaluated and compared with sodium nitroprusside, zaprinast, metformin, and aminoimidazole carboxamide ribonucleotide (AICAR). Methylene blue and L-NAME were used to assess the inhibition of cyclic guanosine monophosphate/nitric oxide pathway. Zinc-protoporphyrin-IX (ZnPP) was also used to investigate the contribution of mevalonate pathway. MAIN OUTCOME MEASURES: The expression of p-AMPK, p-eNOS, AMPK and eNOS induced by beta-lapachone in HUVECs study and the percent relaxation of cavernosal tissue in organ bath study. RESULTS: Beta-lapachone clearly induced AMPK phosphorylation and, as a consequence, eNOS phosphorylation in HUVECs. Beta-lapachone-induced upregulation of eNOS activity was also observed in HUVECs and steadily increased up to 1 hour. In organ bath study, beta-lapachone significantly relaxed the phenylephrine pretreated strips in a dose-dependent manner. This relaxation effect was not totally blocked by methylene blue or L-NAME. After removing endothelium, the relaxation was totally blocked by ZnPP. CONCLUSIONS: A novel AMPK activator, beta-lapachone has a strong relaxation effect on precontracted cavernosal smooth muscle strips in the rabbit. And phosphorylation of AMPK and eNOS strongly related to the action of beta-lapachone. Mevalonate pathway also might be considered as a suggestive mechanism.

Fenofibrate differentially regulates plasminogen activator inhibitor-1 gene expression via adenosine monophosphate-activated protein kinase-dependent induction of orphan nuclear receptor small heterodimer partner.

UNLABELLED: Plasminogen activator inhibitor type I (PAI-1) is a marker of the fibrinolytic system and serves as a possible predictor for hepatic metabolic syndromes. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, is a drug used for treatment of hyperlipidemia. Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of crucial genes involved in various metabolic pathways. In this study, we show that fenofibrate increased SHP gene expression in cultured liver cells and in the normal and diabetic mouse liver by activating the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway in a PPARalpha-independent manner. Administration of transforming growth factor beta (TGF-beta) or a methionine-deficient and choline-deficient (MCD) diet to induce the progressive fibrosing steatohepatitis model in C57BL/6 mice was significantly reversed by fenofibrate via AMPK-mediated induction of SHP gene expression with a dramatic decrease in PAI-1 messenger RNA (mRNA) and protein expression along with other fibrotic marker genes. No reversal was observed in SHP null mice treated with fenofibrate. Treatment with another PPARalpha agonist, WY14643, showed contrasting effects on these marker gene expressions in wild-type and SHP null mice, demonstrating the specificity of fenofibrate in activating AMPK signaling. Fenofibrate exhibited a differential inhibitory pattern on PAI-1 gene expression depending on the transcription factors inhibited by SHP. CONCLUSION: By demonstrating that a PPARalpha-independent fenofibrate-AMPK-SHP regulatory cascade can play a key role in PAI-1 gene down-regulation and reversal of fibrosis, our study suggests that various AMPK activators regulating SHP might provide a novel pharmacologic option in ameliorating hepatic metabolic syndromes.

Expression of a gonadotropin-releasing hormone receptor-simian virus 40 T-antigen transgene has sex-specific effects on the reproductive axis.

The GnRH receptor (GnRHR) responds to pulsatile GnRH signals to coordinate pituitary gonadotropin synthesis and secretion. Previously, a 1.2-kb fragment of the 5'-flanking region isolated from the mouse GnRHR gene was shown to target expression to pituitary gonadotropes in vivo. The 1.2-kb gene promoter fused to the simian virus 40 large T antigen (TAg) was used to generate transgenic mice that form gonadotrope-derived pituitary tumors at 4-5 months of age. Transgenic female mice have hypogonadotropic hypogonadism, infantile gonads, and are infertile throughout their life span, whereas males remain reproductively intact until their tumors become large. We hypothesized that the targeted TAg expression causes a sex-specific disruption of the reproductive axis at the level of the pituitary gland. To test this hypothesis, we characterized the pituitary gonadotropin beta-subunit and TAg expression patterns, and measured plasma gonadotropin and gonadal steroid levels in female and male mice before and after pituitary tumor development. TAg expression was observed in transgenic females and males 15 d of age, before tumor development. Interestingly, and in contrast to the transgenic males, pituitary LH beta and FSH beta subunit protein levels, and plasma LH and FSH levels, were reduced in transgenic females. Reproductive organs in transgenic female mice remained underdeveloped but were normal in transgenic males. We conclude that the expression of the TAg transgene driven by the GnRHR gene promoter results in female-specific infertility due to disruption of gonadotropin production and secretion even before tumor development.

Activation of NAD(P)H:quinone oxidoreductase 1 prevents arterial restenosis by suppressing vascular smooth muscle cell proliferation.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important pathogenic mechanisms in atherosclerosis and restenosis after vascular injury. In this study, we investigated the effects of beta-lapachone (betaL) (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), which is a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase (NQO)1 activity, on neointimal formation in animals given vascular injury and on the proliferation of VSMCs cultured in vitro. betaL significantly reduced the neointimal formation induced by balloon injury. betaL also dose-dependently inhibited the FCS- or platelet-derived growth factor-induced proliferation of VSMCs by inhibiting G(1)/S phase transition. betaL increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 in rat and human VSMCs. Chemical inhibitors of AMPK or dominant-negative AMPK blocked the betaL-induced suppression of cell proliferation and the G(1) cell cycle arrest, in vitro and in vivo. The activation of AMPK in VSMCs by betaL is mediated by LKB1 in the presence of NQO1. Taken together, these results show that betaL inhibits VSMCs proliferation via the NQO1 and LKB1-dependent activation of AMPK. These observations provide the molecular basis that pharmacological stimulation of NQO1 activity is a new therapy for the treatment of vascular restenosis and/or atherosclerosis which are caused by proliferation of VSMCs.

Calcium-sensing receptor stimulates secretion of an interferon-gamma-induced monokine (CXCL10) and monocyte chemoattractant protein-3 in immortalized GnRH neurons.

Biology of GnRH neurons is critically dependent on extracellular Ca(2+) (Ca(2+) (o)). We evaluated differences in gene expression patterns with low and high Ca(2+) (o) in an immortalized GnRH neuron line, GT1-7 cells. Mouse global oligonucleotide microarray was used to evaluate transcriptional differences among the genes regulated by elevated Ca(2+) (o). Our result identified two interferon-gamma (IFNgamma)-inducible chemokines, CXCL9 and CXCL10, and a beta chemokine, monocyte chemoattractant protein-3 (MCP-3/CCL7), being up-regulated in GT1-7 cells treated with high Ca(2+) (o) (3.0 mM) compared with low Ca(2+) (o) (0.5 mM). Up-regulation of these mRNAs by elevated Ca(2+) (o) was confirmed by quantitative PCR. Elevated Ca(2+) (o) stimulated secretion of CXCL10 and MCP-3 but not CXCL9 in GT1-7 cells, and this effect was mediated by an extracellular calcium-sensing receptor (CaR) as the dominant negative CaR attenuated secretion of CXCL10 and MCP-3. CXCL10 and MCP-3 were localized in mouse GnRH neurons in the preoptic hypothalamus. Suppression of K(+) channels (BK channels) with 25 nM charybdotoxin inhibited high-Ca(2+) (o)-stimulated CXCL10 release. Accordingly, CaR activation by a specific CaR agonist, NPS-467, resulted in the activation of a Ca(2+)-activated K(+) channel in these cells. CaR-mediated MCP-3 secretion involves the PI3 kinase pathway in GT1-7 cells. MCP-3 stimulated chemotaxis of astrocytes treated with transforming growth factor-beta (TGFbeta). With TGFbeta-treated astrocytes, we next observed that conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of astrocytes, and this effect was attenuated by a neutralizing antibody to MCP-3. These results implicate CaR as an important regulator of GnRH neuron function in vivo by stimulating secretion of heretofore unsuspected cytokines, i.e., CXCL10 and MCP-3.

Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice.

The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca(2+) concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca(2+)-stimulated chemotaxis. We also demonstrated expression of a beta-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was approximately 27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.

Pituitary homeobox 1 (Pitx1) stimulates rat LHbeta gene expression via two functional DNA-regulatory regions.

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH beta-subunit gene (LHbeta). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHbeta gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHbeta gene promoter. While investigating the role of Pitx1 in regulating rat LHbeta gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions -73 to -52 in the rat LHbeta gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5'Pitx1 site as well as this putative 3'Pitx1 region. In transient transfection analysis, mutation of the LHbeta-3'Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5'Pitx1 site with further loss following mutation of the 3'Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHbeta gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

Oct-1 and nuclear factor Y bind to the SURG-1 element to direct basal and gonadotropin-releasing hormone (GnRH)-stimulated mouse GnRH receptor gene transcription.

The cis-regulatory element localized to position -292/-285 of the mouse GnRH receptor (mGnRHR) gene promoter, designated Sequence Underlying Responsiveness to GnRH 1 (SURG-1), has been shown previously to contribute to stimulation of mGnRHR gene expression by GnRH. We have identified three specific protein-DNA complexes on the SURG-1 element by EMSA using nuclear extracts from the gonadotrope-derived alphaT3-1 and LbetaT2 cell lines. Serial mutagenesis and supershift assays identified nuclear factor Y (NF-Y) binding to -288/-284 and Oct-1 binding to a TAAT sequence at -290/-287. Binding of these two transcription factors was confirmed in vivo by chromatin immunoprecipitation assay and increased in response to GnRH stimulation. To define the functional significance of these sequences in the regulation of mGnRHR gene transcription, transient transfection assays were performed in alphaT3-1 cells using a 1.2-kb mGnRHR (-1164/+62) gene promoter-luciferase reporter construct with selective mutations of the Oct-1, NF-Y, and/or the previously characterized activating protein 1 (AP-1) binding site (-274/-268). Individual mutations in the Oct-1, NF-Y, and AP-1 sites decreased both basal expression and stimulation by GnRH agonist, and the combined mutation of the Oct-1 and AP-1 binding sites further reduced basal transcriptional activity and abolished GnRH stimulation. Overexpression of NF-YA increased GnRHR promoter activity, whereas expression of a dominant negative NF-YA mutant decreased activity, further supporting a role of NF-Y in regulation of mGnRHR gene transcription. In addition, knockdown of Oct-1 by small interfering RNA confirmed that Oct-1 is important for mGnRHR gene expression. In conclusion, NF-Y and Oct-1 bind to the SURG-1 element to direct basal and GnRH-stimulated expression of the mGnRHR gene.

Essential role of the homeodomain for pituitary homeobox 1 activation of mouse gonadotropin-releasing hormone receptor gene expression through interactions with c-Jun and DNA.

The gonadotropin-releasing hormone receptor (GnRHR) is expressed primarily in the gonadotropes of the anterior pituitary. Pituitary homeobox 1 (Pitx-1) has been shown to activate pituitary-specific gene expression by direct DNA binding and/or protein-protein interaction with other transcription factors. We hypothesized that Pitx-1 might also dictate tissue-specific expression of the mouse GnRHR (mGnRHR) gene in a similar manner. Pitx-1 activated the mGnRHR gene promoter, and transactivation was localized to sequences between -308 and -264. Pitx-1 bound to this region only with low affinity. This region includes an activating protein 1 (AP-1) site, which was previously shown to be important for mGnRHR gene expression. Further characterization indicated that an intact AP-1 site was required for full Pitx-1 responsiveness. Furthermore, Pitx-1 and AP-1 were synergistic in the activation of the mGnRHR gene promoter. A Pitx-1 homeodomain (HD) point mutation, which eliminated DNA binding ability, caused only a partial reduction of transactivation, whereas deletion of the HD completely prevented transactivation. Pitx-1 interacted directly with c-Jun, and the HD was sufficient for this interaction. While the point mutation in the Pitx-1 HD did not affect interaction with c-Jun, deletion of the HD eliminated the interaction. Taken together, our studies indicate that Pitx-1 can direct transactivation of the mGnRHR gene, in part by DNA binding and in part by an action of Pitx-1 as a cofactor for AP-1, augmenting AP-1 activity through a novel protein-protein interaction between c-Jun and the HD of Pitx-1.

Impaired leptin expression and abnormal response to fasting in corticotropin-releasing hormone-deficient mice.

Leptin has been postulated to comprise part of an adipostat, whereby during states of excessive energy storage, elevated levels of the hormone prevent further weight gain by inhibiting appetite. A physiological role for leptin in this regard remains unclear because the presence of excessive food, and therefore the need to restrain overeating under natural conditions, is doubtful. We have previously shown that CRH-deficient (Crh(-/-)) mice have glucocorticoid insufficiency and lack the fasting-induced increase in glucocorticoid, a hormone important in stimulating leptin synthesis and secretion. We hypothesized that these mice might have low circulating leptin. Indeed, Crh(-/-) mice exhibited no diurnal variation of leptin, whereas normal littermates showed a clear rhythm, and their leptin levels were lower than their counterparts. A continuous peripheral CRH infusion to Crh(-/-) mice not only restored corticosterone levels, but it also increased leptin expression to normal. Surprisingly, 36 h of fasting elevated leptin levels in Crh(-/-) mice, rather than falling as in normal mice. This abnormal leptin change during fasting in Crh(-/-) mice was corrected by corticosterone replacement. Furthermore, Crh(-/-) mice lost less body weight during 24 h of fasting and ate less food during refeeding than normal littermates. Taken together, we conclude that glucocorticoid insufficiency in Crh(-/-) mice results in impaired leptin production as well as an abnormal increase in leptin during fasting, and propose that the fast-induced physiological reduction in leptin may play an important role to stimulate food intake during the recovery from fasting.