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1.  Muscle-Bound? A Tissue-Selective Nonsteroidal Androgen Receptor Modulator 
Endocrinology  2007;148(1):1-3.
PMCID: PMC4075012  PMID: 17179140
2.  Contribution of endogenous glucocorticoids and their intra-vascular metabolism by 11β-HSDs to post-angioplasty neointimal proliferation in mice 
Endocrinology  2012;153(12):5896-5905.
Exogenous glucocorticoids inhibit neointimal proliferation in animals. We aime to test the hypothesis that endogenous glucocorticoids influence neointimal proliferation; this may be mediated by effects on systemic risk factors or locally in vessels, and modulated either by adrenal secretion or by enzymes expressed in vessels which mediate local inactivation (11β-HSD2 in endothelium) or regeneration (11β-HSD1 in smooth muscle) of glucocorticoids. Femoral artery wire-angioplasty was conducted in C57Bl/6J, Apo-E−/−, 11β-HSD1−/−, Apo-E, 11β-HSD1−/− (double knockout) and 11β-HSD2−/− mice following glucocorticoid administration, adrenalectomy, glucocorticoid or mineralocorticoid receptor antagonism, or selective 11β-HSD1 inhibition. In C57Bl/6J mice, neointimal proliferation was reduced by systemic or local glucocorticoid administration, unaffected by adrenalectomy, reduced by the mineralocorticoid receptor antagonist eplerenone, and increased by the glucocorticoid receptor antagonist RU38486. 11β-HSD2 deletion had no effect on neointimal proliferation, with or without eplerenone. 11β-HSD1 inhibition or deletion had no effect in chow-fed C57Bl/6J mice, but reduced neointimal proliferation in Apo-E−/− mice on Western diet. Reductions in neointimal size were accompanied by reduced macrophage and increased collagen content. We conclude that pharmacological administration of glucocorticoid receptor agonists or of mineralocorticoid receptor antagonists may be useful in reducing neointimal proliferation. Endogenous corticosteroids induce beneficial glucocorticoid receptor activation and adverse mineralocorticoid receptor activation. However, manipulation of glucocorticoid metabolism has beneficial effects only in mice with exaggerated systemic risk factors, suggesting effects mediated primarily in liver and adipose rather than intra-vascular glucocorticoid signalling. Reducing glucocorticoid action with 11β-HSD1 inhibitors that are being developed for type 2 diabetes appears not to risk enhanced neointimal proliferation.
PMCID: PMC3977041  PMID: 23125311
Glucocorticoids; mineralocorticoids; Angioplasty; Neointimal proliferation; 11β-hydroxysteroid dehydrogenases (11β-HSDs)
3.  11β-hydroxysteroid dehydrogenase type 2 deficiency accelerates atherogenesis and causes pro-inflammatory changes in the endothelium in Apoe−/− mice 
Endocrinology  2010;152(1):236-246.
Mineralocorticoid receptor (MR) activation is pro inflammatory and pro atherogenic. Antagonism of MR improves survival in humans with congestive heart failure caused by atherosclerotic disease. In animal models, activation of MR exacerbates atherosclerosis. The enzyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) prevents inappropriate activation of the mineralocorticoid receptor (MR) from inappropriate activation by glucocorticoids by inactivating glucocorticoids in mineralocorticoid-target tissues. To determine whether glucocorticoid-mediated activation of MR increases atheromatous plaque formation we generated Apoe−/−/11β-HSD2−/− double-knockout (E/b2) mice. On chow diet, E/b2 mice developed atherosclerotic lesions by 3 months of age, while Apoe−/− mice remained lesion-free. Brachiocephalic plaques in 3 month-old E/b2 mice showed increased macrophage and lipid content and reduced collagen content compared to similar sized brachiocephalic plaques in 6 month old Apoe−/− mice. Crucially, treatment of E/b2 mice with eplerenone, an MR antagonist, reduced plaque development and macrophage infiltration while increasing collagen and smooth muscle cell content without any effect on systolic blood pressure (SBP). In contrast, reduction of SBP in E/b2 mice using the epithelial sodium channel (ENaC) blocker amiloride produced a less profound atheroprotective effect. Vascular cell adhesion molecule 1 (VCAM-1) expression was increased in the endothelium of E/b2 mice compared to Apoe−/− mice. Similarly, aldosterone increased VCAM-1 expression in mouse aortic endothelial cells, an effect mimicked by corticosterone only in the presence of an 11β-HSD2 inhibitor. Thus, loss of 11β-HSD2 leads to striking atherogenesis associated with activation of MR stimulating pro-inflammatory processes in the endothelium of E/b2 mice.
PMCID: PMC3977042  PMID: 21106873
atherosclerosis; mineralocorticoid receptor; 11β-hydroxysteroid dehydrogenase
Endocrinology  2009;151(1):195-202.
Regeneration of active glucocorticoids within liver and adipose tissue by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) may be of pathophysiological importance in obesity and Metabolic Syndrome and is a therapeutic target in type 2 diabetes. Polymorphisms in HSD11B1, the gene encoding 11β-HSD1, have been associated with metabolic phenotype in humans, including type 2 diabetes and hypertension. Here we have tested the functional consequences of 2 single nucleotide polymorphisms located in contexts that potentially affect tissue levels of 11β-HSD1. We report no effect of allelic variation at rs846910, a polymorphism within the 5′-flanking region of the gene on HSD11B1 promoter activity in vitro. However, compared to the common G allele, the A allele of rs13306421, a polymorphism located 2 nucleotides 5′ to the translation initiation site, gave higher 11β-HSD1 expression and activity in vitro and was translated at higher levels in in vitro translation reactions, possibly associated with a lower frequency of “leaky scanning”. These data suggest that this polymorphism may have direct functional consequences on levels of 11β-HSD1 enzyme activity in vivo. However, the rs13306421 A sequence variant originally reported in other ethnic groups may be of low prevalence as it was not detected in a population of 600 European caucasian women.
PMCID: PMC3971150  PMID: 19934376
steroid metabolism; glucocorticoid; obesity; SNP; translation; regulation
5.  Rapid decreases in preoptic aromatase activity and brain monoamine concentrations after engaging in male sexual behavior 
Endocrinology  2005;146(9):3809-3820.
In Japanese quail as in rats, the expression of male sexual behavior over relatively long time periods (days to weeks) is dependent on the local production of estradiol in the preoptic area via the aromatization of testosterone. On a short-term basis (minutes to hours), central actions of dopamine as well as locally produced estrogens modulate behavioral expression. In rats, a view of and sexual interaction with a female increases dopamine release in the preoptic area. In quail, in vitro brain aromatase activity is rapidly modulated by calcium-dependent phosphorylations that are likely to occur in vivo as a result of changes in neurotransmitter activity. Furthermore, an acute estradiol injection rapidly stimulates copulation in quail, while a single injection of the aromatase inhibitor Vorozole™ rapidly inhibits this behavior. We hypothesized that brain aromatase and dopaminergic activities are regulated in quail in association with the expression of male sexual behavior. Visual access as well as sexual interactions with a female produced a significant decrease in brain aromatase activity that was maximal after 5 min. This expression of sexual behavior also resulted in a significant decrease in dopaminergic as well as serotonergic activity after 1 min, which returned to basal levels after 5 min. These results demonstrate for the first time that aromatase activity is rapidly modulated in vivo in parallel with changes in dopamine activity. Sexual interactions with the female decreased aromatase and dopamine activity. These data challenges established views about the causal relationships among dopamine, estrogen action and male sexual behavior.
PMCID: PMC3909742  PMID: 15932925
male copulatory behavior; aromatization; non-genomic effects; quail; dopamine; serotonin
6.  Leptin-Independent Programming of Adult Body Weight and Adiposity in Mice 
Endocrinology  2011;152(2):10.1210/en.2010-0911.
Low birth weight and rapid postnatal weight gain are independent and additive risk factors for the subsequent development of metabolic disease. Despite an abundance of evidence for these associations, mechanistic data are lacking. The hormone leptin has received significant interest as a potential programming factor, because differences in the profile of leptin in early life have been associated with altered susceptibility to obesity. Whether leptin alone is a critical factor for programming obesity has, until now, remained unclear. Using the leptin-deficient ob/ob mouse, we show that low birth weight followed by rapid catch-up growth during lactation (recuperated offspring) leads to a persistent increase in body weight in adult life, both in wild-type and ob/ob animals. Furthermore, recuperated offspring are hyperphagic and epididymal fat pad weights are significantly increased, reflecting greater adiposity. These results show definitively that factors other than leptin are crucial in the programming of energy homeostasis in this model and are powerful enough to alter adiposity in a genetically obese strain.
PMCID: PMC3884597  PMID: 21209019
7.  The inflammatory response in acyl-CoA oxidase 1 deficiency (pseudoneonatal adrenoleukodystrophy) 
Endocrinology  2012;153(6):2568-2575.
Among several peroxisomal neurodegenerative disorders, the pseudoneonatal adrenoleukodys-trophy (P-NALD) is characterized by the acyl-coenzyme A oxidase 1 (ACOX1) deficiency, which leads to the accumulation of very-long-chain fatty acids (VLCFA) and inflammatory demyelination. However, the components of this inflammatory process in P-NALD remain elusive. In this study, we used transcriptomic profiling and PCR array analyses to explore inflammatory gene expression in patient fibroblasts. Our results show the activation of IL-1 inflammatory pathway accompanied by the increased secretion of two IL-1 target genes, IL-6 and IL-8 cytokines. Human fibroblasts exposed to very-long-chain fatty acids exhibited increased mRNA expression of IL-1α and IL-1β cytokines. Furthermore, expression of IL-6 and IL-8 cytokines in patient fibroblasts was down-regulated by MAPK, p38MAPK, and Jun N-terminal kinase inhibitors. Thus, the absence of acyl-coenzyme A oxidase 1 activity in P-NALD fibroblasts triggers an inflammatory process, in which the IL-1 pathway seems to be central. The use of specific kinase inhibitors may permit the modulation of the enhanced inflammatory status.
PMCID: PMC3791418  PMID: 22508517
Acyl-CoA Oxidase; deficiency; genetics; metabolism; Cells, Cultured; Fatty Acids; pharmacology; Fibroblasts; drug effects; metabolism; pathology; Gene Expression Regulation; drug effects; Humans; Immunohistochemistry; Inflammation; genetics; metabolism; Inflammation Mediators; metabolism; Interleukin-1; genetics; metabolism; Interleukin-6; genetics; metabolism; Interleukin-8; genetics; metabolism; Oligonucleotide Array Sequence Analysis; Osteopontin; genetics; metabolism; Reverse Transcriptase Polymerase Chain Reaction; Transcriptome
8.  Corticotropin-Releasing Hormone Mediates the Response to Cold Stress in the Neonatal Rat without Compensatory Enhancement of the Peptide’s Gene Expression* 
Endocrinology  1994;135(6):2364-2368.
A variety of stressors activate the hypothalamic-pituitary-adrenal axis, with secretion and compensatory enhanced synthesis of hypothalamic corticotropin-releasing hormone (CRH). Whether CRH is a major effector in the stress response of the neonatal rat and whether the peptide's gene expression is subsequently up-regulated are not fully understood. We studied the effect of cold-separation stress on plasma corticosterone (CORT) levels and CRH messenger RNA (CRH-mRNA) abundance in the paraventricular nucleus. Rats (4–16 days old) were subjected to maximal tolerated cold-separation. CORT and CRH-mRNA abundance were measured before and at several time points after stress. Cold-separation stress resulted in a significant plasma CORT increase in all age groups studied. This was abolished by the administration of an antiserum to CRH on both postnatal days 6 and 9. CRH-mRNA increased in rats aged 9 days or older, but not in 6-day-old rats, by 4 h after stress. These results suggest the presence of robust CRH-mediated adrenal responses to cold-separation stress in neonatal rats. Before postnatal day 9, however, the compensatory increase in CRH-mRNA abundance is minimal.
PMCID: PMC3783019  PMID: 7988418
9.  Quantitative Trait Loci Associated with Elevated Thyroid-Stimulating Hormone in the Wistar-Kyoto Rat 
Endocrinology  2004;146(2):870-878.
Thyroid hormones are essential for the regulation of developmental and physiological processes. The genetic factors underlying naturally occurring variability in mammalian thyroid function are, however, only partially understood. Genetic control of thyroid function can be studied with animal models such as the inbred Wistar-Kyoto (WKY) rat strain. Previous studies established that WKY rats have elevated TSH, slightly elevated total T3, and normal total T4 levels compared with Wistar controls. The present study confirmed a persistent 24-h elevation of TSH in WKY rats compared with the Fisher 344 (F344) rat, another inbred strain. Acute T3 challenge (25 g/ 100 g body weight ip) suppressed serum TSH and T4 levels in both strains. Quantitative trait locus analysis of elevated TSH in a reciprocally bred WKY × F344 F2 population identified one highly significant locus on chromosome 6 (LOD = 11.7, TSH-1) and one suggestive locus on chromosome 5 (LOD = 2.3, TSH-2). The confidence interval of TSH-1 contains the TSH receptor and type 2 deiodinase genes, and TSH-2 contains the type 1 deiodinase gene. The WKY alleles of each gene contain sequence alterations, but additional studies are indicated to identify the specific gene or genes responsible for altered regulation of the thyroid axis. These findings suggest that one or more genetic alterations within the TSH-1 locus significantly contribute to the altered thyroid function tests of the WKY rat.
PMCID: PMC3764449  PMID: 15514085
10.  Rapid Inhibition of Neurons in the Dorsal Motor Nucleus of the Vagus by Leptin 
Endocrinology  2006;148(4):1868-1881.
The peptide leptin conveys the availability of adipose energy stores to the brain. Increasing evidence implicates a significant role for extrahypothalamic sites of leptin action, including the dorsal vagal complex, a region critical for regulating visceral parasympathetic function. The hypothesis that leptin suppresses cellular activity in the dorsal motor nucleus of the vagus nerve (DMV) was tested using whole-cell patch-clamp recordings in brainstem slices. Leptin caused a rapid membrane hyperpolarization in 50% of rat DMV neurons. Leptin also hyperpolarized a subset of gastric-related neurons (62%), identified after gastric inoculation with a transneuronal retrograde viral tracer. The hyperpolarization was associated with a decrease in input resistance and cellular responsiveness and displayed characteristics consistent with an increased K+ conductance. Perfusion of tolbutamide (200 μM) reversed the leptin-induced hyperpolarization, and tolbutamide or wortmannin (10–100 nM) prevented the hyperpolarization, indicating that leptin activated an ATP-sensitive K+ channel via a phosphoinositide-3-kinase-dependent mechanism. Leptin reduced the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs), whereas inhibitory postsynaptic currents (IPSCs) were largely unaffected. Electrical stimulation of the nucleus tractus solitarii (NTS) resulted in constant-latency EPSCs, which were decreased in amplitude by leptin. The paired-pulse ratio was increased, suggesting leptin effects involved activation of receptors presynaptic to the recorded neuron. A leptin-induced suppression of EPSCs, but not IPSCs, evoked by focal photolytic uncaging of glutamate within the NTS was also observed, supportive of leptin effects on the glutamatergic NTS projection to the DMV. Therefore, leptin directly hyperpolarized and indirectly suppressed excitatory synaptic activity to DMV neurons involved in visceral regulation, including gastric-related neurons.
PMCID: PMC3761087  PMID: 17194747
11.  A Rapid Release of Corticosteroid-Binding Globulin from the Liver Restrains the Glucocorticoid Hormone Response to Acute Stress 
Endocrinology  2011;152(10):3738-3748.
A strict control of glucocorticoid hormone responses to stress is essential for health. In blood, glucocorticoid hormones are for the largest part bound to corticosteroid-binding globulin (CBG), and just a minor fraction of hormone is free. Only free glucocorticoid hormone is able to exert biological effects, but little is known about its regulation during stress. We found, using a dual-probe in vivo microdialysis method, that in rats, the forced-swim stress-induced rise in free corticosterone (its major glucocorticoid hormone) is strikingly similar in the blood and in target compartments such as the subcutaneous tissue and the brain. However, in all compartments, the free corticosterone response was delayed by 20–30 min as compared with the total corticosterone response in the blood. We discovered that CBG is the key player in this delay. Swim stress evoked a fast (within 5 min) and profound rise in CBG protein and binding capacity in the blood through a release of the protein from the liver. Thus, the increase in circulating CBG levels after stress restrains the rise in free corticosterone concentrations for approximately 20 min in the face of mounting total hormone levels in the circulation. The stress-induced increase in CBG seems to be specific for moderate and strong stressors. Both restraint stress and forced swimming caused an increase in circulating CBG, whereas its levels were not affected by mild novelty stress. Our data uncover a new, highly dynamic role for CBG in the regulation of glucocorticoid hormone physiology after acute stress.
PMCID: PMC3722548  PMID: 21828178
12.  The Melanocortinergic Pathway Is Rapidly Recruited by Emotional Stress and Contributes to Stress-Induced Anorexia and Anxiety-Like Behavior 
Endocrinology  2007;148(11):5531-5540.
Neurons producing melanocortin receptor agonist, α-MSH derived from proopiomelanocortin, and antagonist, agouti-related protein, are known to be sensitive to metabolic stress such as food deprivation and glucoprivation. However, how these neurons respond to emotional/psychological stress remained to be elucidated. We report here that acute emotional stressors, i.e. restraint and forced swim, evoked mRNA expression of c-fos, a neuronal activation marker, in a high percentage of proopiomelanocortin neurons (up to 53% for restraint stress and 62% for forced swim), with marked variations along the rostro-caudal axis of the arcuate nucleus. In contrast, only a small population of agouti-related protein neurons in this brain region was activated. These neuronal activation patterns were correlated with behavioral reactions. Both stressors suppressed feeding and induced anxiety-like behavior in the elevated plus-maze test, as reflected by a reduction in the percentage of entries and time spent in the open arms. Central pretreatment with SHU9119, a melanocortin receptor antagonist, dose dependently attenuated the anorectic and anxiogenic effects elicited by acute restraint or forced swim. These results indicate that the melancortinergic pathway can be rapidly recruited by acute emotional stress, and that activation of melanocortin signaling is involved in mediating stress-induced anorexia and anxiety.
PMCID: PMC3708592  PMID: 17673512
13.  [No title available] 
PMCID: PMC3639543  PMID: 22234470
14.  Disruption of the Murine Glp2r Impairs Paneth Cell Function and Increases Susceptibility to Small Bowel Enteritis 
Endocrinology  2012;153(3):1141-1151.
Exogenous glucagon-like peptide-2 receptor (GLP-2R) activation elicits proliferative and cytoprotective responses in the gastrointestinal mucosa and ameliorates experimental small and large bowel gut injury. Nevertheless, the essential physiological role(s) of the endogenous GLP-2R remain poorly understood. We studied the importance of the GLP-2R for gut growth, epithelial cell lineage allocation, the response to mucosal injury, and host-bacterial interactions in Glp2r−/− and littermate control Glp2r+/+ mice. Glp2r−/− mice exhibit normal somatic growth and preserved small and large bowel responses to IGF-I and keratinocyte growth factor. However, Glp2r−/− mice failed to up-regulate intestinal epithelial c-fos expression in response to acute GLP-2 administration and do not exhibit changes in small bowel conductance or small or large bowel growth after administration of GLP-2R agonists. The crypt and villus compartment and the numbers and localization of Paneth, enteroendocrine, and goblet cells were comparable in Glp2r+/+ vs. Glp2r−/− mice. Although the severity and extent of colonic mucosal injury in response to 3% oral dextran sulfate was similar across Glp2r genotypes, Glp2r−/− mice exhibited significantly increased morbidity and mortality and increased bacterial translocation after induction of enteritis with indomethacin and enhanced mucosal injury in response to irinotecan. Moreover, bacterial colonization of the small bowel was significantly increased, expression of Paneth cell antimicrobial gene products was reduced, and mucosal bactericidal activity was impaired in Glp2r−/− mice. Although the Glp2r is dispensable for gut development and the response to colonic injury, Glp2r−/− mice exhibit enhanced sensitivity to small bowel injury, and abnormal host-bacterial interactions in the small bowel.
PMCID: PMC3606134  PMID: 22253424 CAMSID: cams2742
15.  Phosphorylation of Human Insulin Receptor Substrate-1 at Serine 629 Plays a Positive Role in Insulin Signaling 
Endocrinology  2007;148(10):4895-4905.
The function of insulin receptor substrate-1 (IRS-1) is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, whereas phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of human IRS-1 at Ser629 was increased by insulin in Chinese hamster ovary cells expressing the insulin receptor (1.26 ± 0.09-fold; P < 0.05) and L6 cells (1.35 ± 0.29-fold; P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser629 revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser629 in cells was decreased upon treatment with either an Akt inhibitor or by coexpression with kinase dead Akt, whereas Ser629 phosphorylation was increased by coexpression with constitutively active Akt. In addition, Ser629 of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser629 by a Ser629Ala mutation resulted in increased phosphorylation of Ser636, a known negative regulator of IRS-1, without affecting phosphorylation of Tyr632 or Ser616. Cells expressing the Ser629Ala mutation, along with increased Ser636 phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol 3′-kinase with IRS-1 and decreased phosphorylation of Akt at Ser473. Finally, in vitro phosphorylation of a Ser629-containing IRS-1 fragment with Akt reduces the subsequent ability of ERK to phosphorylate Ser636/639. These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser629, resulting in decreased phosphorylation of IRS-1 at Ser636 and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and, ultimately, the pathogenesis of type 2 diabetes.
PMCID: PMC3581341  PMID: 17640984
16.  Sex-Dependent Effects of Neonatal Inflammation on Adult Inflammatory Markers and Behavior 
Endocrinology  2010;151(6):2689-2699.
Inflammatory molecules, such as cyclooxygenase (COX), a prostaglandin synthetic enzyme, have been identified as a marker of depressive symptomology. Previously, we have observed elevated basal COX-2 expression in the hypothalamus of adult male rats treated neonatally with lipopolysaccharide (LPS), which might suggest a phenotype for disrupted hedonic behavior, a symptom of depression. However, COX-2 and its contribution to the expression of anhedonic behavior has not been investigated in these males or in female rats across the estrous cycle, which is the purpose of the current work. Here, we examine the effects of a neonatal LPS challenge or saline on the sucrose preference test as a measure of anhedonia, and hypothalamic COX-2 expression, in adult male and freely cycling female rats. Our data indicate a sex difference in that neonatal LPS at postnatal d 14 causes elevated basal expression of hypothalamic COX-2 in male, but not in female, rats. Additionally, baseline sucrose preference in male and female rats was unaltered as a function of neonatal LPS treatment or estrous cycle stage. In both male and female animals, 50 μg/kg LPS in adulthood caused elevated plasma IL-6 and hypothalamic COX-2 expression in neonatally saline-treated rats but significantly less so in neonatally LPS-treated rats of both sexes; this neonatal programming was not evident for sucrose preference or for total fluid intake (even after much higher doses of LPS). Our data are suggestive of a dissociation between inflammation and anhedonic behavior and a differential effect of neonatal inflammation in males and females.
PMCID: PMC3524265  PMID: 20392837 CAMSID: cams2587
17.  GnRH neurons directly listen to the periphery 
Endocrinology  2011;152(10):3589-3591.
PMCID: PMC3519581  PMID: 21937749
Animals; Blood-Brain Barrier; Dendrites; physiology; Female; Gonadotropin-Releasing Hormone; physiology; Hypothalamus; physiology
18.  Neurochemical Characterization of Body Weight-Regulating Leptin Receptor Neurons in the Nucleus of the Solitary Tract 
Endocrinology  2012;153(10):4600-4607.
The action of peripherally released leptin at long-form leptin receptors (LepRb) within the brain represents a fundamental axis in the regulation of energy homeostasis and body weight. Efforts to delineate the neuronal mediators of leptin action have recently focused on extrahypothalamic populations and have revealed that leptin action within the nucleus of the solitary tract (NTS) is critical for normal appetite and body weight regulation. To elucidate the neuronal circuits that mediate leptin action within the NTS, we employed multiple transgenic reporter lines to characterize the neurochemical identity of LepRb-expressing NTS neurons. LepRb expression was not detected in energy balance-associated NTS neurons that express cocaine- and amphetamine-regulated transcript, brain-derived neurotrophic factor, neuropeptide Y, nesfatin, catecholamines, γ-aminobutyric acid, prolactin-releasing peptide, or nitric oxide synthase. The population of LepRb-expressing NTS neurons was comprised of subpopulations marked by a proopiomelanocortin-enhanced green fluorescent protein (EGFP) transgene and distinct populations that express proglucagon and/or cholecystokinin. The significance of leptin action on these three populations of NTS neurons was assessed in leptin-deficient Ob/Ob mice, revealing increased NTS proglucagon and cholecystokinin, but not proopiomelanocortin, expression. These data provide new insight into the appetitive brainstem circuits engaged by leptin.
PMCID: PMC3507354  PMID: 22869346
19.  Exendin-4 as a Stimulator of Rat Insulin I Gene Promoter Activity via bZIP/CRE Interactions Sensitive to Serine/Threonine Protein Kinase Inhibitor Ro 31-8220 
Endocrinology  2002;143(6):2303-2313.
Signal transduction properties of exendin-4 (Ex-4) underlying its ability to stimulate rat insulin I gene promoter (RIP1) activity were assessed in the pancreatic β-cell line INS-1. Ex-4 acted via glucagon-like peptide-1 receptors to stimulate RIP1 in a glucose-dependent manner, as measured in cells transfected with a −410-bp RIP1-luciferase construct (RIP1-Luc). The action of Ex-4 was independent of cAMP and PKA because it was not blocked by cotransfection with dominant-negative Gαs, was unaffected by pretreatment with the membrane-permeant cAMP antagonist 8-Br-Rp-cAMPS, and remained apparent after treatment with PKA inhibitors H-89 or KT 5720. Similarly, cotransfection with a dominant-negative isoform of the type-2 cAMP-regulated guanine nucleotide exchange factor (Epac2) failed to alter the response to Ex-4. Ro 31-8220, a serine/threonine protein kinase inhibitor that targets PKC as as well as the 90-kDa ribosomal S6 kinase (RSK) and mitogenand stress-activated protein kinase (MSK) family of cAMP response element-binding protein (CREB) kinases, blocked the stimulatory action of Ex-4 at RIP1-Luc. However, selective inhibition of PKC using K-252c, prolonged exposure to phorbol 1,2-myristate-13-acetate, or cotransfection with dominant-negative atypical PKC-ζ, was without effect. A-CREB, a dominant-negative inhibitor of basic region-leucine zipper transcription factors (bZIPs) related in structure to CREB, inhibited the action of Ex-4 at RIP1-Luc, whereas A-ATF-2 was ineffective. Similarly, introduction of deletions at the RIP1 cAMP response element (CRE), or truncation of RIP1 to remove the CRE, nearly abolished the action of Ex-4. Inactivating mutations introduced at the A4/A3 elements, binding sites for the glucose-regulated homeodomain transcription factor PDX-1, did not diminish the response to Ex-4, although a marked reduction of basal promoter activity was observed. The glucose-dependent stimulation of RIP1-Luc by Ex-4 was reproduced using a synthetic reporter (RIP1-CRE-Luc) incorporating multimerized CREs of the RIP1 nonpalindromic sequence 5′-TGACGTCC-3′. It is concluded that the bZIP and CRE-mediated stimulation of RIP1 by Ex-4 explains, at least in part, how this insulinotropic hormone facilitates transcriptional activity of the rat insulin I gene.
PMCID: PMC3500996  PMID: 12021195
20.  Increased Expression of CYR61, an Extracellular Matrix Signaling Protein, in Human Benign Prostatic Hyperplasia and Its Regulation by Lysophosphatidic Acid 
Endocrinology  2004;145(6):2929-2940.
Lysophosphatidic acid (LPA) is an endogenous lipid growth factor that is thought to play important roles in cell proliferation and antiapoptosis and therefore may have roles in the development and progression of benign prostatic hyperplasia (BPH). CYR61 (CCN1), on the other hand, is a growth factor-inducible immediate early gene that functions in cell proliferation, differentiation, and extracellular matrix synthesis. Here we show the close relationship between LPA-induced expression of CYR61 and prostate enlargement. CYR61 mRNA and protein were dramatically up-regulated by 18:1 LPA (oleoyl-LPA) within 1 and 2 h, respectively, in both stromal and epithelial prostatic cells. G protein-coupled receptors, i.e. Edg-2, Edg-4, and Edg-7, for LPA were also expressed in both stromal and epithelial prostatic cells. Furthermore, on DNA microarray analysis for normal and BPH patients, CYR61 was found to be related to the development and progression of BPH, regardless of symptoms. Although CYR61 mRNA was synthesized in hyperplastic epithelial cells, in many cases of BPH, CYR61 protein was detected in both the epithelial and stromal regions of BPH patient tissues. The functional contribution of CYR61 to prostatic cell growth was demonstrated by recombinant CYR61 protein and anti-CYR61 neutralizing antibodies, which inhibited CYR61-dependent cell spreading and significantly diminished cell proliferation, respectively. In conclusion, these data support the hypothesis that LPAs induce the expression of CYR61 by activating G protein-coupled receptors and that CYR61 acts as a secreted autocrine and/or paracrine mediator in stromal and epithelial hyperplasia, demonstrating the potential importance of this signaling mechanism in the disease. (Endocrinology 145: 2929–2940, 2004)
PMCID: PMC3477642  PMID: 14988385
21.  Lipopolysaccharide initiates inflammation in bovine granulosa cells via the TLR4 pathway and perturbs oocyte meiotic progression in vitro 
Endocrinology  2011;152(12):5029-5040.
Infections of the reproductive tract or mammary gland with Gram-negative bacteria perturb ovarian function, follicular growth and fecundity in cattle. We hypothesised that lipopolysaccharide (LPS) from Gram-negative bacteria stimulates an inflammatory response by ovarian granulosa cells that is mediated by TLR4. The present study tested the capacity of bovine ovarian granulosa cells to initiate an inflammatory response to pathogen associated molecular patterns (PAMPs), and determined subsequent effects on the in vitro maturation of oocytes. Granulosa cells elicited an inflammatory response to PAMPs (LPS, lipoteichoic acid, peptidoglycan or Pam3CSK4) with accumulation of the cytokine IL-6, and the chemokine IL-8, in a time- and dose-dependent manner. Granulosa cells responded acutely to LPS with rapid phosphorylation of TLR signaling components, p38 and ERK, and increased expression of IL6 and IL8 mRNA, although nuclear translocation of p65 was not evident. Targeting TLR4 with siRNA, attenuated granulosa cell accumulation of IL-6 in response to LPS. Endocrine function of granulosa cells is regulated by FSH, but here FSH also enhanced responsiveness to LPS, increasing IL-6 and IL-8 accumulation. Furthermore, LPS stimulated IL-6 secretion and expansion by cumulus-oocyte complexes (COCs), and increased rates of meiotic arrest and germinal vesicle breakdown failure. In conclusion, bovine granulosa cells initiate an innate immune response to LPS via the TLR4 pathway leading to inflammation and to perturbation of meiotic competence.
PMCID: PMC3428914  PMID: 21990308
Granulosa cell; immunity; inflammation; lipopolysaccharide; oocyte maturation; ovary; Toll-like receptor 4
22.  Minireview: The Neuroendocrinology of the Suprachiasmatic Nucleus as a Conductor of Body Time in Mammals 
Endocrinology  2007;148(12):5640-5647.
Circadian rhythms in physiology and behavior are regulated by a master clock resident in the suprachiasmatic nucleus (SCN) of the hypothalamus, and dysfunctions in the circadian system can lead to serious health effects. This paper reviews the organization of the SCN as the brain clock, how it regulates gonadal hormone secretion, and how androgens modulate aspects of circadian behavior known to be regulated by the SCN. We show that androgen receptors are restricted to a core SCN region that receives photic input as well as afferents from arousal systems in the brain. We suggest that androgens modulate circadian behavior directly via actions on the SCN and that both androgens and estrogens modulate circadian rhythms through an indirect route, by affecting overall activity and arousal levels. Thus, this system has multiple levels of regulation; the SCN regulates circadian rhythms in gonadal hormone secretion, and hormones feed back to influence SCN functions.
PMCID: PMC3423957  PMID: 17901227
23.  Neurochemical Characterization of Body Weight-Regulating Leptin Receptor Neurons in the Nucleus of the Solitary Tract 
Endocrinology  2012;153(10):4600-4607.
The action of peripherally released leptin at long-form leptin receptors (LepRb) within the brain represents a fundamental axis in the regulation of energy homeostasis and body weight. Efforts to delineate the neuronal mediators of leptin action have recently focused on extrahypothalamic populations and have revealed that leptin action within the nucleus of the solitary tract (NTS) is critical for normal appetite and body weight regulation. To elucidate the neuronal circuits that mediate leptin action within the NTS, we employed multiple transgenic reporter lines to characterize the neurochemical identity of LepRb-expressing NTS neurons. LepRb expression was not detected in energy balance-associated NTS neurons that express cocaine- and amphetamine-regulated transcript, brain-derived neurotrophic factor, neuropeptide Y, nesfatin, catecholamines, γ-aminobutyric acid, prolactin-releasing peptide, or nitric oxide synthase. The population of LepRb-expressing NTS neurons was comprised of subpopulations marked by a proopiomelanocortin-enhanced green fluorescent protein (EGFP) transgene and distinct populations that express proglucagon and/or cholecystokinin. The significance of leptin action on these three populations of NTS neurons was assessed in leptin-deficient Ob/Ob mice, revealing increased NTS proglucagon and cholecystokinin, but not proopiomelanocortin, expression. These data provide new insight into the appetitive brainstem circuits engaged by leptin.
PMCID: PMC3507354  PMID: 22869346
24.  Corticotropin releasing factor receptor type II (CRF2) messenger ribonucleic acid levels in the hypothalamic ventromedial nucleus of the infant rat are reduced by maternal deprivation 
Endocrinology  1997;138(11):5048-5051.
The stress neurohormone corticotropin releasing factor (CRF) activates at least two receptor types. Expression of corticotropin releasing factor receptor type II (CRF2) has been demonstrated in the hypothalamic ventromedial nucleus (VMH) of the adult and developing rat, but the physiological functions of VMH-CRF2 have not been elucidated. The VMH has been documented as an important participant in the regulation of food intake and its interactions with the hypothalamic-pituitary-adrenal axis and circadian rhythms. Regulation of VMH-CRF2 may thus play a role in the interplay of physiological alterations in metabolic state with the neuroendocrine and anorexic effects of CRF. This study determined the regulation of CRF2-mRNA expression in infant rats by the physiological consequences of maternal deprivation, i.e., fasting and stress. Using in situ hybridization, maternally deprived pups had an average 62% reduction of VMH-CRF2-mRNA levels compared with stress-free controls. Maternal deprivation also resulted in elevated plasma corticosterone levels (3.8 ± 0.3 vs. 1.3 ± 0.1μg/dl) and an average 5.7% body weight loss. This study demonstrates that maternal deprivation, via fasting and HPA activation, leads to a dramatic decrease of CRF2-mRNA levels in the VMH. These results are consistent with a role for CRF2 activation in mediating some of the complex interactions of CRF (or urocortin) with regulation of food intake in the developing rat.
PMCID: PMC3404504  PMID: 9348237
25.  Neuropeptide Y Cells Represent a Distinct Glucose-Sensing Population in the Lateral Hypothalamus 
Endocrinology  2011;152(11):4046-4052.
The maintenance of appropriate glucose levels is necessary for survival. Within the brain, specialized neurons detect glucose fluctuations and alter their electrical activity. These glucose-sensing cells include hypothalamic arcuate nucleus neurons expressing neuropeptide Y (NPY) and lateral hypothalamic area (LHA) neurons expressing orexin/hypocretins (ORX) or melanin-concentrating hormone (MCH). Within the LHA, a population of NPY-expressing cells exists; however, their ability to monitor energy status is unknown. We investigated whether NPY neurons located in the LHA, a classic hunger center, detect and respond to fluctuations in glucose availability and compared these responses with those of known LHA glucose sensors expressing ORX or MCH. Using mice expressing green fluorescent protein under the control of NPY regulatory elements, we identified LHA NPY cells and explored their anatomical distribution, neurochemical and electrical properties, in vivo responses to fasting and insulin-induced hypoglycemia, and in situ electrical responses to extracellular glucose. We report that NPY, ORX, and MCH are expressed in nonoverlapping populations within the LHA. Subpopulations of LHA NPY neurons were activated in vivo by both a 6-h fast and insulin-induced hypoglycemia. Likewise, increased extracellular glucose suppressed the electrical activity of approximately 70% of LHA NPY neurons in situ, eliciting hyperpolarization and activating background K+ currents. Furthermore, we report that the glucose sensitivity of LHA NPY neurons is significantly different from neighboring ORX and MCH neurons. These data suggest that NPY-expressing cells in the LHA are a novel population of glucose-sensing neurons that represent a new player in the brain circuitry integrating information about glucose homeostasis.
PMCID: PMC3328128  PMID: 21914773

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