Leukemia inhibitory factor suppresses hepatic de novo lipogenesis and induces cachexia in mice.

Cancer cachexia is a systemic metabolic syndrome characterized by involuntary weight loss, and muscle and adipose tissue wasting. Mechanisms underlying cachexia remain poorly understood. Leukemia inhibitory factor (LIF), a multi-functional cytokine, has been suggested as a cachexia-inducing factor. In a transgenic mouse model with conditional LIF expression, systemic elevation of LIF induces cachexia. LIF overexpression decreases de novo lipogenesis and disrupts lipid homeostasis in the liver. Liver-specific LIF receptor knockout attenuates LIF-induced cachexia, suggesting that LIF-induced functional changes in the liver contribute to cachexia. Mechanistically, LIF overexpression activates STAT3 to downregulate PPARα, a master regulator of lipid metabolism, leading to the downregulation of a group of PPARα target genes involved in lipogenesis and decreased lipogenesis in the liver. Activating PPARα by fenofibrate, a PPARα agonist, restores lipid homeostasis in the liver and inhibits LIF-induced cachexia. These results provide valuable insights into cachexia, which may help develop strategies to treat cancer cachexia.

Blocking AMPKαS496 phosphorylation improves mitochondrial dynamics and hyperglycemia in aging and obesity.

Impaired mitochondrial dynamics causes aging-related or metabolic diseases. Yet, the molecular mechanism responsible for the impairment of mitochondrial dynamics is still not well understood. Here, we report that elevated blood insulin and/or glucagon levels downregulate mitochondrial fission through directly phosphorylating AMPKα at S496 by AKT or PKA, resulting in the impairment of AMPK-MFF-DRP1 signaling and mitochondrial dynamics and activity. Since there are significantly increased AMPKα1 phosphorylation at S496 in the liver of elderly mice, obese mice, and obese patients, we, therefore, designed AMPK-specific targeting peptides (Pa496m and Pa496h) to block AMPKα1S496 phosphorylation and found that these targeting peptides can increase AMPK kinase activity, augment mitochondrial fission and oxidation, and reduce ROS, leading to the rejuvenation of mitochondria. Furthermore, these AMPK targeting peptides robustly suppress liver glucose production in obese mice. Our data suggest these targeting peptides are promising therapeutic agents for improving mitochondrial dynamics and activity and alleviating hyperglycemia in elderly and obese patients.

Gluconeogenesis Flux in Metabolic Disease.

Gluconeogenesis is a critical biosynthetic process that helps maintain whole-body glucose homeostasis and becomes altered in certain medical diseases. We review gluconeogenic flux in various medical diseases, including common metabolic disorders, hormonal imbalances, specific inborn genetic errors, and cancer. We discuss how the altered gluconeogenic activity contributes to disease pathogenesis using data from experiments using isotopic tracer and spectroscopy methodologies. These in vitro, animal, and human studies provide insights into the changes in circulating levels of available gluconeogenesis substrates and the efficiency of converting those substrates to glucose by gluconeogenic organs. We highlight ongoing knowledge gaps, discuss emerging research areas, and suggest future investigations. A better understanding of altered gluconeogenesis flux may ultimately identify novel and targeted treatment strategies for such diseases.

Activation of the canonical ER stress IRE1-XBP1 pathway by insulin regulates glucose and lipid metabolism.

Knockout of the transcription factor X-box binding protein (XBP1) is known to decrease liver glucose production and lipogenesis. However, whether insulin can regulate gluconeogenesis and lipogenesis through XBP1 and how insulin activates the inositol-requiring enzyme-XBP1 ER stress pathway remains unexplored. Here, we report that in the fed state, insulin-activated kinase AKT directly phosphorylates inositol-requiring enzyme 1 at S724, which in turn mediates the splicing of XBP1u mRNA, thus favoring the generation of the spliced form, XBP1s, in the liver of mice. Subsequently, XBP1s stimulate the expression of lipogenic genes and upregulates liver lipogenesis as previously reported. Intriguingly, we find that fasting leads to an increase in XBP1u along with a drastic decrease in XBP1s in the liver of mice, and XBP1u, not XBP1s, significantly increases PKA-stimulated CRE reporter activity in cultured hepatocytes. Furthermore, we demonstrate that overexpression of XBP1u significantly increases cAMP-stimulated expression of rate-limiting gluconeogenic genes, G6pc and Pck1, and glucose production in primary hepatocytes. Reexpression of XBP1u in the liver of mice with XBP1 depletion significantly increases fasting blood glucose levels and gluconeogenic gene expression. These data support an important role of XBP1u in upregulating gluconeogenesis in the fasted state. Taken together, we reveal that insulin signaling via AKT controls the expression of XBP1 isoforms and that XBP1u and XBP1s function in different nutritional states to regulate liver gluconeogenesis and lipogenesis, respectively.

Targeting hepatic kisspeptin receptor ameliorates nonalcoholic fatty liver disease in a mouse model.

Nonalcoholic fatty liver disease (NAFLD), the most common liver disease, has become a silent worldwide pandemic. The incidence of NAFLD correlates with the rise in obesity, type 2 diabetes, and metabolic syndrome. A hallmark featureof NAFLD is excessive hepatic fat accumulation or steatosis, due to dysregulated hepatic fat metabolism, which can progress to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Currently, there are no approved pharmacotherapies to treat this disease. Here, we have found that activation of the kisspeptin 1 receptor (KISS1R) signaling pathway has therapeutic effects in NAFLD. Using high-fat diet-fed mice, we demonstrated that a deletion of hepatic Kiss1r exacerbated hepatic steatosis. In contrast, enhanced stimulation of KISS1R protected against steatosis in wild-type C57BL/6J mice and decreased fibrosis using a diet-induced mouse model of NASH. Mechanistically, we found that hepatic KISS1R signaling activates the master energy regulator, AMPK, to thereby decrease lipogenesis and progression to NASH. In patients with NAFLD and in high-fat diet-fed mice, hepatic KISS1/KISS1R expression and plasma kisspeptin levels were elevated, suggesting a compensatory mechanism to reduce triglyceride synthesis. These findings establish KISS1R as a therapeutic target to treat NASH.

Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays.

The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFß inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays.

A Direct Comparison of Thyroid Hormone Receptor Protein Levels in Mice Provides Unexpected Insights into Thyroid Hormone Action.

Background: Thyroid hormone (TH) action is mediated by three major thyroid hormone receptor (THR) isoforms α1, ß1, and ß2 (THRA1, THRB1, and THRB2). These THRs and a fourth major but non-TH binding isoform, THRA2, are encoded by two genes Thra and Thrb. Reliable antibodies against all THR isoforms are not available, and THR isoform protein levels in mammalian tissues are often inferred from messenger RNA (mRNA) levels. Methods: We generated knock-in mouse models expressing endogenously and identically 2X hemagglutenin epitope (HA)-tagged THRs (THRA1/2, THRB1, and THRB2), which could then be detected by commercially available anti-HA antibodies. Using nuclear enrichment, immunoprecipitation, and Western blotting, we determined relative THR protein expression in 16 mouse organs. Results: In all peripheral organs tested except the liver, the predominant THR isoform was THRA1. Surprisingly, in metabolically active organs such as fat and muscle, THRB1 protein levels were up to 10 times lower than that of THRA1, while their mRNA levels appeared similar. In contrast to peripheral organs, the central nervous system (CNS) had a unique pattern with relatively low levels of both THRB1 and THRA1, and high levels of THRA2 expression. As expected, THRB2 was highly expressed in the pituitary, but a previously unknown sex-specific difference in THRB2 expression was found (female mice having higher pituitary expression than male mice). Higher THRB2 expression appears to make the central axis more sensitive to TH as both serum thyrotropin and Tshb mRNA levels were lower in female mice. Conclusions: Direct comparison of THR protein abundance in different organs using endogenously tagged HA-THR mouse lines shows that expression of THR isoforms is regulated at transcriptional and posttranscriptional levels, and in organ-specific manner. The prevalence of THRA1 and low abundance of THRB1 in majority of peripheral tissues suggest that peripheral actions of these isoforms should be revisited. A unique pattern of high THRA2 in CNS warrants further exploration of this non-TH binding isoform in brain development. Finally, THRB2, in addition to cell-specific control, is also regulated in a sex-specific manner, which may change the hypothalamus-pituitary-thyroid axis set point and perhaps metabolism in males and females.

In-Source CID Ramping and Covariant Ion Analysis of Hydrophilic Interaction Chromatography Metabolomics.

A large proportion of the complexity and redundancy of LC-MS metabolomics data comes from adduct formation. To reduce such redundancy, many tools have been developed to recognize and annotate adduct ions. These tools rely on predefined adduct lists that are generated empirically from reversed-phase LC-MS studies. In addition, hydrophilic interaction chromatography (HILIC) is gaining popularity in metabolomics studies due to its enhanced performance over other methods for polar compounds. HILIC methods typically use high concentrations of buffer salts to improve chromatographic performance. Therefore, it is necessary to analyze adduct formation in HILIC metabolomics. To this end, we developed covariant ion analysis (COVINA) to investigate metabolite adduct formation. Using this tool, we completely annotated 201 adduct and fragment ions from 10 metabolites. Many of the metabolite adduct ions were found to contain cluster ions corresponding to mobile phase additives. We further utilized COVINA to find the major ionized forms of metabolites. Our results show that for some metabolites, the adduct ion signals can be >200-fold higher than the signals from the deprotonated form, offering better sensitivity for targeted metabolomics analysis. Finally, we developed an in-source CID ramping (InCIDR) method to analyze the intensity changes of the adduct and fragment ions from metabolites. Our analysis demonstrates a promising method to distinguish the protonated and deprotonated ions of metabolites from the adduct and fragment ions.

Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK.

Impaired mitochondrial respiratory activity contributes to the development of insulin resistance in type 2 diabetes. Metformin, a first-line antidiabetic drug, functions mainly by improving patients' hyperglycemia and insulin resistance. However, its mechanism of action is still not well understood. We show here that pharmacological metformin concentration increases mitochondrial respiration, membrane potential, and ATP levels in hepatocytes and a clinically relevant metformin dose increases liver mitochondrial density and complex 1 activity along with improved hyperglycemia in high-fat- diet (HFD)-fed mice. Metformin, functioning through 5' AMP-activated protein kinase (AMPK), promotes mitochondrial fission to improve mitochondrial respiration and restore the mitochondrial life cycle. Furthermore, HFD-fed-mice with liver-specific knockout of AMPKα1/2 subunits exhibit higher blood glucose levels when treated with metformin. Our results demonstrate that activation of AMPK by metformin improves mitochondrial respiration and hyperglycemia in obesity. We also found that supra-pharmacological metformin concentrations reduce adenine nucleotides, resulting in the halt of mitochondrial respiration. These findings suggest a mechanism for metformin's anti-tumor effects.

Impairments in the reproductive axis of female mice lacking estrogen receptor ß in GnRH neurons.

The effect of estrogen on the differentiation and maintenance of reproductive tissues is mediated by two nuclear estrogen receptors (ERs), ERα, and ERß. Lack of functional ERα and ERß genes in vivo significantly affects reproductive function; however, the target tissues and signaling pathways in the hypothalamus are not clearly defined. Here, we describe the generation and reproductive characterization of a complete-ERß KO (CERßKO) and a GnRH neuron-specific ERßKO (GERßKO) mouse models. Both ERßKO mouse models displayed a delay in vaginal opening and first estrus. Hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression levels in both ERßKO mice were similar to control mice; however female CERßKO and GERßKO mice had lower basal and surge serum gonadotropin levels. Although a GnRH stimulation test in both female ERßKO models showed preserved gonadotropic function in the same animals, a kisspeptin stimulation test revealed an attenuated response by GnRH neurons, suggesting a role for ERß in normal GnRH neuron function. No alteration in estrogen-negative feedback was observed in either ERßKO mouse models after ovariectomy and estrogen replacement. Further, abnormal development of ovarian follicles with low serum estradiol levels and impairment of fertility were observed in both ERßKO mouse models. In male ERßKO mice, no differences in the timing of pubertal onset or serum luteinizing hormone and follicle-stimulating hormone levels were observed as compared with controls. Taken together, these data provide in vivo evidence for a role of ERß in GnRH neurons in modulating puberty and reproduction, specifically through kisspeptin responsiveness in the female hypothalamic-pituitary-gonadal axis.

Activation of the cAMP-PKA pathway Antagonizes Metformin Suppression of Hepatic Glucose Production.

Metformin is the most commonly prescribed oral anti-diabetic agent worldwide. Surprisingly, about 35% of diabetic patients either lack or have a delayed response to metformin treatment, and many patients become less responsive to metformin over time. It remains unknown how metformin resistance or insensitivity occurs. Recently, we found that therapeutic metformin concentrations suppressed glucose production in primary hepatocytes through AMPK; activation of the cAMP-PKA pathway negatively regulates AMPK activity by phosphorylating AMPKα subunit at Ser-485, which in turn reduces AMPK activity. In this study, we find that metformin failed to suppress glucose production in primary hepatocytes with constitutively activated PKA and did not improve hyperglycemia in mice with hyperglucagonemia. Expression of the AMPKα1(S485A) mutant, which is unable to be phosphorylated by PKA, increased both AMPKα activation and the suppression of glucose production in primary hepatocytes treated with metformin. Intriguingly, salicylate/aspirin prevents the phosphorylation of AMPKα at Ser-485, blocks cAMP-PKA negative regulation of AMPK, and improves metformin resistance. We propose that aspirin/salicylate may augment metformin's hepatic action to suppress glucose production.

Altered somatotroph feedback regulation improves metabolic efficiency and limits adipose deposition in male mice.

Several transgenic mouse models with disruption in the growth hormone (GH) axis support the role of GH in augmenting metabolic homeostasis. Specifically, interest has focused on GH's lipolytic properties and ability to affect adipose deposition. Furthermore, both GH and insulin growth factor 1 (IGF-1) may also play a direct or indirect role in adipose development. The somatotroph insulin-like growth factor-1 receptor knockout (SIGFRKO) mouse with only a modest increase in serum GH and IGF-1 demonstrates less adipose tissue than controls. In order to characterize the metabolic phenotype of SIGFRKO mice, histologic analysis of fat depots confirmed a smaller average diameter of adipocytes in the SIGFRKO mice compared to controls. These changes were accompanied by an increase in lipolytic gene expression in fat depots. Indirect calorimetry performed on 6-8week old male mice and again at 25weeks of age demonstrated that SIGFRKO mice, at both ages, had a higher VO2 and increased energy expenditure when compared with controls. The calculated respiratory exchange ratio (RER) was lower in the younger SIGFRKO mice compared to controls. No differences in food consumption or in either ambulatory or total activity were seen between SIGFRKO and control mice in either age group. These studies highlight the role of GH in adipose deposition and its influence on the expression of lipolytic genes resulting in an altered metabolic state, thus providing a mechanism for the decrease in weight gain seen in the SIGFRKO mouse model.

Metformin Acts on Two Different Molecular Pathways to Enhance Adult Neural Precursor Proliferation/Self-Renewal and Differentiation.

The recruitment of endogenous adult neural stem cells for brain repair is a promising regenerative therapeutic strategy. This strategy involves stimulation of multiple stages of adult neural stem cell development, including proliferation, self-renewal, and differentiation. Currently, there is a lack of a single therapeutic approach that can act on these multiple stages of adult neural stem cell development to enhance neural regeneration. Here we show that metformin, an FDA-approved diabetes drug, promotes proliferation, self-renewal, and differentiation of adult neural precursors (NPCs). Specifically, we show that metformin enhances adult NPC proliferation and self-renewal dependent upon the p53 family member and transcription factor TAp73, while it promotes neuronal differentiation of these cells by activating the AMPK-aPKC-CBP pathway. Thus, metformin represents an optimal candidate neuro-regenerative agent that is capable of not only expanding the adult NPC population but also subsequently driving them toward neuronal differentiation by activating two distinct molecular pathways.

Insulin receptor signaling in the GnRH neuron plays a role in the abnormal GnRH pulsatility of obese female mice.

Infertility associated with obesity is characterized by abnormal hormone release from reproductive tissues in the hypothalamus, pituitary, and ovary. These tissues maintain insulin sensitivity upon peripheral insulin resistance. Insulin receptor signaling may play a role in the dysregulation of gonadotropin-releasing hormone (GnRH) secretion in obesity, but the interdependence of hormone secretion in the reproductive axis and the multi-hormone and tissue dysfunction in obesity hinders investigations of putative contributing factors to the disrupted GnRH secretion. To determine the role of GnRH insulin receptor signaling in the dysregulation of GnRH secretion in obesity, we created murine models of diet-induced obesity (DIO) with and without intact insulin signaling in the GnRH neuron. Obese control female mice were infertile with higher luteinizing hormone levels and higher GnRH pulse amplitude and total pulsatile secretion compared to lean control mice. In contrast, DIO mice with a GnRH specific knockout of insulin receptor had improved fertility, luteinizing hormone levels approaching lean mice, and GnRH pulse amplitude and total secretion similar to lean mice. Pituitary responsiveness was similar between genotypes. These results suggest that in the obese state, insulin receptor signaling in GnRH neurons increases GnRH pulsatile secretion and consequent LH secretion, contributing to reproductive dysfunction.

Developmental and endocrine regulation of kisspeptin expression in mouse Leydig cells.

Kisspeptin, encoded by the Kiss1 gene, binds to a specific G protein-coupled receptor (kisspeptin1 receptor) to regulate the central reproductive axis. Kisspeptin has also been reported to be expressed in peripheral tissues, including the testes. However, factors regulating testicular kisspeptin and its role in reproduction are unknown. Our objective herein was to begin to address kisspeptin function in the testis. In particular, we sought to determine the level of kisspeptin in the testis in comparison with the brain and other tissues, how these levels change from the prepubertal period through sexual maturation, and the factors involved in kisspeptin regulation in the testis. Immunohistochemical analysis of testis sections using a validated kisspeptin antibody localized kisspeptin to the Leydig cells. Kisspeptin was not detected in germ cells or Sertoli cells within the seminiferous tubules at any developmental time period studied, from prepuberty to sexual maturation. A developmental time course of testicular kisspeptin revealed that its mRNA and protein levels increased during development, reaching robust levels at postnatal day 28, correlating with pubertal onset. In vitro studies of primary mouse Leydig cells, as well as in vivo studies, indicated clearly that LH is involved in regulating levels of Leydig cell kisspeptin. Interestingly, gonadectomy resulted in elevated LH but reduced serum kisspeptin levels, suggesting that testicular kisspeptin may be secreted. These data document kisspeptin expression in mouse Leydig cells, its secretion into peripheral serum, and its regulation by changes in reproductive neuroendocrine function.

Metformin activates AMP-activated protein kinase by promoting formation of the αßγ heterotrimeric complex.

Metformin is the most widely prescribed oral anti-diabetic agent. Recently, we have shown that low metformin concentrations found in the portal vein suppress glucose production in hepatocytes through activation of AMPK. Moreover, low concentrations of metformin were found to activate AMPK by increasing the phosphorylation of AMPKα at Thr-172. However, the mechanism underlying the increase in AMPKα phosphorylation at Thr-172 and activation by metformin remains unknown. In the current study, we find that low concentrations of metformin promote the formation of the AMPK αßγ complex, resulting in an increase in net phosphorylation of the AMPK α catalytic subunit at Thr-172 by augmenting phosphorylation by LKB1 and antagonizing dephosphorylation by PP2C.

Low concentrations of metformin suppress glucose production in hepatocytes through AMP-activated protein kinase (AMPK).

Metformin is a first-line antidiabetic agent taken by 150 million people across the world every year, yet its mechanism remains only partially understood and controversial. It was proposed that suppression of glucose production in hepatocytes by metformin is AMPK-independent; however, unachievably high concentrations of metformin were employed in these studies. In the current study, we find that metformin, via an AMP-activated protein kinase (AMPK)-dependent mechanism, suppresses glucose production and gluconeogenic gene expression in primary hepatocytes at concentrations found in the portal vein of animals (60-80 µM). Metformin also inhibits gluconeogenic gene expression in the liver of mice administered orally with metformin. Furthermore, the cAMP-PKA pathway negatively regulates AMPK activity through phosphorylation at Ser-485/497 on the α subunit, which in turn reduces net phosphorylation at Thr-172. Because diabetic patients often have hyperglucagonemia, AMPKα phosphorylation at Ser-485/497 is a therapeutic target to improve metformin efficacy.

Circadian regulation of Tshb gene expression by Rev-Erbα (NR1D1) and nuclear corepressor 1 (NCOR1).

Thyroid hormones (TH) are critical for development, growth, and metabolism. Circulating TH levels are tightly regulated by thyroid-stimulating hormone (TSH) secretion within the hypothalamic-pituitary-thyroid axis. Although circadian TSH secretion has been well documented, the mechanism of this observation remains unclear. Recently, the nuclear corepressor, NCOR1, has been postulated to regulate TSH expression, presumably by interacting with thyroid hormone receptors (THRs) bound to TSH subunit genes. We report herein the first in vitro study of NCOR1 regulation of TSH in a physiologically relevant cell system, the TαT1.1 mouse thyrotroph cell line. Knockdown of NCOR1 by shRNA adenovirus increased baseline Tshb mRNA levels compared with scrambled control, but surprisingly had no affect on the T3-mediated repression of this gene. Using ChIP, we show that NCOR1 enriches on the Tshb promoter at sites different from THR previously identified by our group. Furthermore, NCOR1 enrichment on Tshb is unaffected by T3 treatment. Given that NCOR1 does not target THR on Tshb, we hypothesized that NCOR1 targeted Rev-Erbα (NR1D1), an orphan nuclear receptor that is a potent repressor of gene transcription and regulator of metabolism and circadian rhythms. Using a serum shock technique, we synchronized TαT1.1 cells to study circadian gene expression. Post-synchronization, Tshb and Nr1d1 mRNA levels displayed oscillations that inversely correlated with each other. Furthermore, NR1D1 was enriched at the same locus as NCOR1 on Tshb. Therefore, we propose a model for Tshb regulation whereby NR1D1 and NCOR1 interact to regulate circadian expression of Tshb independent of TH negative regulation.

Obesity-induced infertility and hyperandrogenism are corrected by deletion of the insulin receptor in the ovarian theca cell.

Women with polycystic ovary syndrome (PCOS) exhibit elevated androgen levels, oligoanovulation, infertility, and insulin resistance in metabolic tissues. The aims of these studies were to determine the role of insulin signaling in the development and function of ovarian theca cells and the pathophysiologic effects of hyperinsulinism on ovarian function in obesity. We disrupted the insulin receptor (IR) gene specifically in the theca-interstitial (TI) cells of the ovaries (Cyp17IRKO). No changes in reproductive development or function were observed in lean Cyp17IRKO female mice, suggesting that insulin signaling in TI cell is not essential for reproduction. However, when females were fed a high-fat diet, diet-induced obesity (DIO) wild-type (DIO-WT) mice were infertile and experienced increased circulating testosterone levels, whereas DIO-Cyp17IRKO mice exhibited improved fertility and testosterone levels comparable to those found in lean mice. The levels of phosphorylated IRS1 and CYP17 protein were higher in the ovary of DIO-WT compared with DIO-Cyp17IRKO or lean mice. Ex vivo studies using a whole ovary culture model demonstrated that insulin acts independently or additively with human chorionic gonadotropin to enhance androstenedione secretion. These studies reveal the causal pathway linking hyperinsulinism with ovarian hyperandrogenism and the infertility of obesity.

Both estrogen receptor α and ß stimulate pituitary GH gene expression.

Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α- and ERß-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERß mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERß in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.