Sharon L. Wardlaw

The hypothalamic control of the menstrual cycle and the role of endogenous opioid peptides

Publisher Summary This chapter presents a study involving a rhesus monkey that demonstrates changes in the arcuate oscillator caused by ovarian steroids during the menstrual cycle. The chapter discusses the role of endogenous opioid peptides in the central modulation of gonadotropin secretion by ovarian steroids. The modulation of gonadotropin-releasing hormone (GnRH) and consequently luteinizing hormone (LH) pulsatile secretion by ovarian steroids involves to a certain degree an interaction with the hypothalamic opioid peptide network. β-endorphin secretion into the hypophyseal portal circulation—a reflection of hypothalamic β-endorphin secretory activity—fluctuates cyclically and in accordance with the endocrine milieu. The stimulatory action of naloxone on GnRH and LH release parallels that of endogenous opioid secretion. The sequential acceleration and deceleration of the GnRH oscillator during the menstrual cycle, brought about by cyclic β-endorphin variations, are important determinants of menstrual cyclicity as experimental disturbance of this sequence results in acyclicity for a prolonged period of time.

The effect of running on plasma β-endorphin

Abstract Plasma β-endorphin immunoactivity was measured by RIA in 26 trained long distance runners on 35 occasions before and after running. Mean total β-endorphin immunoactivity increased from 11.8 ± 1.8 (SEM) to 17.6 ± 3.1 pg/m1 in 20 runners after an easy run ( p = .067), and from 8.2 ± 1.03 to 28.0 ± 6.3 pg/ m 1 in 15 runners after a strenuous run (p = .008). Total β-endorphin immunoactivity in the plasma extracts of 7 runners before and after the strenuous run was further characterized by Sephadex G-50 chromatography in order to separate β-endorphin from corssreacting β-lipotropin (β-LPH). A rise in β-endorphin and β-LPH concentrations after running was noted in 5 out of 7 runners.

Lymphocytic hypophysitis. Report of 3 new cases and review of the literature.

Lymphocytic hypophysitis is an uncommon but increasingly recognized disorder characterized by chronic inflammation and destruction of the anterior pituitary. Three new cases are presented here with a review of the 27 previously reported cases. The disease affects primarily young women in late pregnancy or in the postpartum period but also has been described in postmenopausal women and in one man. It presents as an expanding intrasellar mass or as partial or panhypopituitarism. The etiology may be autoimmune. The natural history of this entity begins with enlargement of the pituitary secondary to inflammatory infiltration and progresses to atrophy of the gland with destruction of pituitary tissue and replacement with fibrosis. At least 1 patient had documented recovery of pituitary function, and the overall potential incidence of recoverable function is unknown. Our improved understanding of this disease has led us to conclude that surgical intervention is not always necessary.

Leptin Regulation of Agrp and Npy mRNA in the Rat Hypothalamus

Agouti‐related protein (AGRP) is synthesized in the same neurones in the arcuate nucleus as neuropeptide Y (NPY), another potent orexigenic peptide. AGRP antagonizes the action of α‐melanocyte stimulating hormone, a derivative of pro‐opiomelanocortin (POMC) at the hypothalamic MC4 receptor to increase food intake. Although leptin has been shown to regulate Agrp/Npy and Pomc‐expressing neurones, there are differences with respect to Agrp regulation in leptin receptor‐deficient mice and rats. Unlike the obese leptin receptor‐deficient db/db mouse, which exhibits upregulation of Agrp mRNA expression in the medial basal hypothalamus (MBH) compared to lean controls, the obese leptin receptor‐deficient (faf; Koletsky) rat does not exhibit upregulation of Agrp expression. To determine whether this represents a general difference between leptin receptor‐deficient mice and rats, neuropeptide gene expression was analysed in the MBH of lean and obese rats segregating for a different leptin receptor mutation, Leprfa (Zucker). Fasting in lean rats (+/fa) for 72 h significantly increased Agrp and Npy mRNA expression, and decreased Pomc mRNA expression as detected by a sensitive solution hybridization/S1 nuclease protection assay. Npy mRNA levels were significantly increased in fed obese fa/fa compared to lean rats, and further increased in the obese animals after fasting. In contrast, Agrp mRNA levels did not differ between fed lean and fed obese rats, and fasting did not significantly change Agrp levels in obese rats. To determine whether the change in Agrp expression that occurs with food deprivation in lean rats could be prevented by leptin replacement, Sprague‐Dawley rats were fasted and infused via subcutaneous osmotic micropumps for 48 h with either saline or recombinant mouse leptin. Fasting significantly increased Agrp and Npy, and decreased Pomc mRNA levels. Leptin infusion almost completely reversed these changes such that there was no significant difference between the levels in the fasted rats and those that were fed ad libitum. Thus, in fasted lean rats, Agrp and Npy are upregulated in parallel when leptin levels fall and are downregulated by leptin infusion. By contrast, the absence of a functional leptin receptor results in the upregulation of Npy but not Agrp mRNA.

Effects of sex steroids on brain β-endorphin

Abstract Brain β-endorphin was measured by radioimmunoassay in female rats during different stages of the estrous cycle, during pregnancy, 3 weeks after ovariectomy, and 3 weeks after ovariectomy plus estradiol or estradiol and progesterone replacement. No change in hypothalamic β-endorphin content was noted on the afternoon of diestrus, proestrus, or estrus. However, in 9 rats studied between days 8–20 of pregnancy the mean hypothalamic β-endorphin concentration of41.6 ± 2.24ng/mg protein was significantly higher than the concentration of32.7 ± 1.01 in 21 non-pregnant animals (P We conclude that physiologic concentrations of estradiol and progesterone can alter the content of brain β-endorphin and suggest that ovarian steroids may be important regulators of this brain peptide.

Partially redundant enhancers cooperatively maintain Mammalian pomc expression above a critical functional threshold.

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases.

Effects of adrenalectomy on AGRP, POMC, NPY and CART gene expression in the basal hypothalamus of fed and fasted rats.

Glucocorticoids regulate body energy balance through both peripheral and central mechanisms. In order to understand the central mechanisms that mediate these effects of glucocorticoids we studied the effects of adrenalectomy (ADX) and food deprivation on the expression of four neuropeptide genes (measured by S1 nuclease protection assay) in the medial basal hypothalamus (MBH), which are known to regulate energy balance: pro-opiomelanocortin (POMC), agouti-related peptide (AGRP), neuropeptide Y (NPY), and cocaine and amphetamine regulated transcript (CART). Adult male rats were ADX or sham operated (SHAM), and studied 1-2 weeks later. In the first study effects of ADX and corticosterone replacement on POMC and AGRP expression were determined. ADX decreased POMC and AGRP gene expression in the MBH by 27 and 38%, respectively, compared to SHAM rats. Corticosterone treatment increased the expression of POMC by 87% and AGRP by 45% in ADX rats. The second study was designed to determine if glucocorticoids are necessary for the fasting induced changes in POMC, AGRP, NPY and CART in the MBH. ADX caused a 20-30% decrease in the expression of all four neuropeptide genes in the MBH. As expected, fasting suppressed POMC and CART expression and increased AGRP and NPY expression. The fasting-induced increases in AGRP and NPY persisted after ADX but no further significant decreases in POMC or CART were noted after fasting in ADX rats. Plasma leptin and insulin declined significantly after ADX and increased with corticosterone replacement; both leptin and insulin declined further in fasted, ADX animals. In conclusion, ADX decreases both anorexigenic, POMC and CART, and orexigenic, AGRP and NPY, neuropeptide gene expression in the MBH. AGRP and NPY decrease after ADX despite the fall in plasma leptin and insulin concentrations which in other situations would increase these neuropeptides. Furthermore, glucocorticoids are not required for fasting-induced upregulation of AGRP and NPY expression.

Prolylcarboxypeptidase regulates food intake by inactivating α-MSH in rodents

The anorexigenic neuromodulator alpha-melanocyte-stimulating hormone (alpha-MSH; referred to here as alpha-MSH1-13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of alpha-MSH1-13 maturation and inactivation is incompletely understood. Here we have provided insight into alpha-MSH1-13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of alpha-MSH1-13, producing alpha-MSH1-12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where alpha-MSH1-13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet-induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active alpha-MSH1-13 levels.

The obesity susceptibility gene Cpe links FoxO1 signaling in hypothalamic pro-opiomelanocortin neurons with regulation of food intake

Reduced food intake brings about an adaptive decrease in energy expenditure that contributes to the recidivism of obesity after weight loss. Insulin and leptin inhibit food intake through actions in the central nervous system that are partly mediated by the transcription factor FoxO1. We show that FoxO1 ablation in pro-opiomelanocortin (Pomc)-expressing neurons in mice (here called Pomc-Foxo1−/− mice) increases Carboxypeptidase E (Cpe) expression, resulting in selective increases of α-melanocyte–stimulating hormone (α-Msh) and carboxy-cleaved β-endorphin, the products of Cpe-dependent processing of Pomc. This neuropeptide profile is associated with decreased food intake and normal energy expenditure in Pomc-Foxo1−/− mice. We show that Cpe expression is downregulated by diet-induced obesity and that FoxO1 deletion offsets the decrease, protecting against weight gain. Moreover, moderate Cpe overexpression in the arcuate nucleus phenocopies features of the FoxO1 mutation. The dissociation of food intake from energy expenditure in Pomc-Foxo1−/− mice represents a model for therapeutic intervention in obesity and raises the possibility of targeting Cpe to develop weight loss medications.

FoxO1 Target Gpr17 Activates AgRP Neurons to Regulate Food Intake

Hypothalamic neurons expressing Agouti-related peptide (AgRP) are critical for initiating food intake, but druggable biochemical pathways that control this response remain elusive. Thus, genetic ablation of insulin or leptin signaling in AgRP neurons is predicted to reduce satiety but fails to do so. FoxO1 is a shared mediator of both pathways, and its inhibition is required to induce satiety. Accordingly, FoxO1 ablation in AgRP neurons of mice results in reduced food intake, leanness, improved glucose homeostasis, and increased sensitivity to insulin and leptin. Expression profiling of flow-sorted FoxO1-deficient AgRP neurons identifies G-protein-coupled receptor Gpr17 as a FoxO1 target whose expression is regulated by nutritional status. Intracerebroventricular injection of Gpr17 agonists induces food intake, whereas Gpr17 antagonist cangrelor curtails it. These effects are absent in Agrp-Foxo1 knockouts, suggesting that pharmacological modulation of this pathway has therapeutic potential to treat obesity.